CN116380856A

CN116380856A - Transient stimulated Raman excitation fluorescence spectrum method and system

Info

Publication number: CN116380856A
Application number: CN202310316379.6A
Authority: CN
Inventors: 熊汗青; 姚政见; 余乔智
Original assignee: Peking University
Current assignee: Peking University
Priority date: 2023-03-28
Filing date: 2023-03-28
Publication date: 2023-07-04

Abstract

The invention relates to the technical field of spectrum detection and optical imaging, and provides a transient stimulated Raman excitation fluorescence spectrum method and a system, wherein the method comprises the steps of generating two stimulated Raman excitation pulse pairs with relative delay and adjustable relative delay based on the stimulated Raman excitation pulse pairs; based on the two stimulated Raman excitation pulse pairs, exciting a sample to be detected to generate transient stimulated Raman scattering, and based on the detection pulse, exciting the sample to be detected to generate a fluorescent signal; and collecting a fluorescent signal generated by the sample to be detected, and carrying out Fourier transform on the fluorescent signal to obtain a Raman spectrum corresponding to the sample to be detected. Because two stimulated Raman excitation pulse pairs with adjustable phase-to-phase delay are introduced, a sample to be detected is excited to generate transient stimulated Raman scattering, so that the sample to be detected generates a fluorescent signal containing Raman resonance frequency in a time domain when being excited by detection pulses, and further a clear Raman spectrum without fluorescent background is seen in the frequency domain after Fourier transformation, and the quality of the Raman spectrum is ensured.

Description

Transient stimulated Raman excitation fluorescence spectrum method and system

Technical Field

The invention relates to the technical field of spectrum detection and optical imaging, in particular to a transient stimulated Raman excitation fluorescence spectrum method and system.

Background

Raman spectroscopy is a powerful tool for analyzing molecular structures, can contain abundant sample molecular structure information, can be used for analyzing the dynamics of sample molecules and the interaction with solvent environment, and is widely applied to the research of molecular reaction processes such as biology, chemistry and the like. However, the scattering cross section of raman spectrum is very small and the detection sensitivity is low. And is disturbed by a fluorescent background.

Based on this, stimulated raman excitation fluorescence (Stimulated Raman Excited Fluorescence, SREF) spectroscopic techniques have evolved. However, the raman spectrum obtained by the SREF spectrum technology has a strong and inherent fluorescence background, and the existing excitation technology can not effectively inhibit the fluorescence background.

Based on this, it is urgently needed to provide a transient stimulated raman excitation fluorescence spectroscopy method.

Disclosure of Invention

The invention provides a transient stimulated Raman excitation fluorescence spectrum method and system, which are used for solving the defect that the existing excitation technology in the prior art cannot effectively inhibit the fluorescence background.

The invention provides a transient stimulated Raman excitation fluorescence spectrum method, which comprises the following steps:

generating two stimulated raman excitation pulse pairs with relative delays and adjustable relative delays based on the stimulated raman excitation pulse pairs; the stimulated raman excitation pulse pair comprises a pump pulse and a stokes pulse which are generated based on femtosecond pulse laser and are aligned in time dimension and space dimension;

Based on the two stimulated Raman excitation pulse pairs, exciting a sample to be detected to generate transient stimulated Raman scattering, and based on detection pulses, exciting the sample to be detected to generate a fluorescent signal;

and collecting the fluorescent signal, and carrying out Fourier transform on the fluorescent signal to obtain a Raman spectrum corresponding to the sample to be detected.

According to the transient stimulated Raman excitation fluorescence spectrum method provided by the invention, the femtosecond pulse laser is generated based on a femtosecond laser;

and degeneracy information exists between the stimulated Raman excitation pulse pair and the detection pulse, and the degeneracy information is determined based on the bandwidth of the femtosecond laser, the Raman shift of the stimulated Raman mode of the sample to be detected and the energy level structure.

According to the method for transient stimulated raman excitation fluorescence spectrum provided by the invention, fourier transformation is carried out on the fluorescence signal to obtain a raman spectrum corresponding to the sample to be detected, and the method comprises the following steps:

filtering the light background with the wavelength except the fluorescence signal, and selecting out fluorescence photons at the focal position in the fluorescence signal;

and recording a time domain fluorescence signal corresponding to the fluorescence photon, and carrying out Fourier transform on the time domain fluorescence signal to obtain the Raman spectrum.

According to the transient stimulated raman excitation fluorescence spectrum method provided by the invention, the method for exciting the sample to be detected to generate transient stimulated raman scattering based on two stimulated raman excitation pulse pairs comprises the following steps:

and adjusting the bandwidth of the Stokes pulse so that the difference of the bandwidths of the Stokes pulse and the pump pulse is within a preset range.

The invention provides a transient stimulated Raman excitation fluorescence spectrum system, which comprises: a femtosecond laser light source, a delay scanning device and a signal acquisition device;

the femtosecond laser light source is used for generating stimulated Raman excitation pulse pairs and detection pulses, wherein the stimulated Raman excitation pulse pairs comprise pump pulses and Stokes pulses which are generated based on femtosecond pulse laser and are aligned in time dimension and space dimension;

the time delay scanning device is used for generating two stimulated Raman excitation pulse pairs with relative delay and adjustable relative delay based on the stimulated Raman excitation pulse pairs, wherein the two stimulated Raman excitation pulse pairs are used for exciting a sample to be detected to generate transient stimulated Raman scattering so that the sample to be detected is excited by the detection pulse to generate a fluorescent signal;

The signal acquisition device is used for acquiring the fluorescent signal and carrying out Fourier transform on the fluorescent signal to obtain a Raman spectrum corresponding to the sample to be detected.

According to the transient stimulated Raman excitation fluorescence spectrum system provided by the invention, the femtosecond laser light source comprises a femtosecond laser, and the femtosecond laser is used for generating the femtosecond pulse laser;

degenerate 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 to be measured, and the energy level structure.

According to the transient stimulated raman excitation fluorescence spectrum system provided by the invention, no degeneracy exists between the stimulated raman excitation pulse pair and the detection pulse, or the pump pulse and the detection pulse are degenerated; in response to this, the control unit,

the femtosecond laser light source comprises 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 for generating the femtosecond pulse laser; the polarization beam splitter is used for splitting the femtosecond pulse laser into a first partial beam and a second partial beam;

The first light source branch comprises a first dispersion compensation device, and the first dispersion compensation device is used for carrying out dispersion compensation on the first partial light beam to obtain the Stokes pulse;

the second light source branch comprises a frequency multiplier, an optical parametric oscillator, a second dispersion compensation device and an adjustable first optical delay line; the frequency multiplier is used for multiplying the frequency of the second partial light beam to obtain frequency-multiplied laser; the optical parametric oscillator is driven by the frequency multiplication laser and outputs the pumping pulse; the first optical delay line is configured to align the pump pulse with the stokes pulse in a time dimension; the second dispersion compensation device is used for performing dispersion compensation on the pump pulse;

the first dichroic mirror is for aligning the pump pulse with the stokes pulse in a spatial dimension.

According to the transient stimulated Raman excitation fluorescence spectrum system provided by the invention, the first light source branch further comprises a spectrum filtering device;

the spectrum filtering device is used for adjusting the bandwidth of the Stokes pulse so that the difference of the bandwidth of the Stokes pulse and the bandwidth of the pumping pulse is within a preset range.

According to the transient stimulated raman excitation fluorescence spectrum system provided by the invention, degeneracy exists between the stimulated raman excitation pulse pair and the detection pulse; in response to this, the control unit,

the femtosecond laser light source further comprises a detection laser, a second optical delay line and a second dichroic mirror, wherein the second optical delay line and the second dichroic mirror are arranged along the optical path;

the detection laser is used for generating the detection pulse;

the second optical delay line is used for controlling delay existing between the detection pulse and a target pulse pair, wherein the target pulse pair is a stimulated Raman excitation pulse pair with two stimulated Raman excitation pulse pairs with later time;

the second dichroic mirror is used to align the two stimulated raman excitation pulse pairs with the detection pulse in a spatial dimension.

According to the transient stimulated raman excitation fluorescence spectrum system provided by the invention, the time delay scanning device comprises an interferometer, and one arm of the interferometer comprises an adjustable third optical delay line.

According to the transient stimulated Raman excitation fluorescence spectrum system provided by the invention, the third optical delay line is fixed on the displacement table;

the signal acquisition device is triggered based on the position information of the displacement table.

According to the transient stimulated Raman excitation fluorescence spectrum system provided by the invention, the interferometer further comprises a first beam splitter, a second beam splitter and a reference signal detection device;

the first beam splitter is used for splitting a target beam of the stimulated Raman excitation pulse pair generated by the femtosecond laser light source into a third partial beam and a fourth partial beam, the third partial beam is transmitted along a first arm of the interferometer, and the fourth partial beam is transmitted along a second arm of the interferometer;

the second beam splitter is used for combining the light beam output by the first arm and the light beam output by the second arm, and respectively obtaining a fifth part of light beam and a sixth part of light beam, wherein the fifth part of light beam is used for exciting the sample to be detected to generate transient stimulated Raman scattering and the fluorescent signal;

the reference signal detection device is used for receiving the sixth partial light beam, converting the sixth partial light beam into a reference signal with the coherence time larger than a preset threshold value, and calibrating the position information of the displacement table.

According to the transient stimulated Raman excitation fluorescence spectrum system provided by the invention, the reference signal detection device comprises a grating, a lens and a slit;

And the sixth part of light beams sequentially pass through the grating, the lens and the slit to obtain the reference signal.

According to the transient stimulated Raman excitation fluorescence spectrum system provided by the invention, the signal acquisition device comprises an optical filter, a screening element, a photoelectric detector and a data acquisition card, wherein the optical filter, the screening element, the photoelectric detector and the data acquisition card are arranged along an optical path;

the optical filter is used for filtering out light background with wavelengths except the fluorescent signal;

the screening element is used for selecting fluorescent photons at a focal position in the fluorescent signal;

the photoelectric detector is used for recording a time domain fluorescence signal corresponding to the fluorescence photon;

the data acquisition card is used for carrying out Fourier transform on the time domain fluorescent signals to obtain the Raman spectrum.

The invention provides a transient stimulated Raman excitation fluorescence spectrum method and a system, wherein the method comprises the following steps: generating two stimulated raman excitation pulse pairs with relative delays and adjustable relative delays based on the stimulated raman excitation pulse pairs; based on the two stimulated Raman excitation pulse pairs, exciting a sample to be detected to generate transient stimulated Raman scattering, and based on the detection pulse, exciting the sample to be detected to generate a fluorescent signal; and collecting a fluorescence signal, and carrying out Fourier transform on the fluorescence signal to obtain a Raman spectrum corresponding to the sample to be detected. Because two stimulated Raman excitation pulse pairs with adjustable phase-to-phase delay are introduced, a sample to be detected is excited to generate transient stimulated Raman scattering, so that the sample to be detected can generate a fluorescent signal containing Raman resonance frequency in the time domain when being excited by detection pulses, and further clear Raman spectrum without fluorescence background can be seen in the frequency domain after Fourier transformation, the quality of the Raman spectrum can be ensured, and the Raman spectrum is prevented from being influenced by the fluorescence background.

Drawings

In order to more clearly illustrate the invention or the technical solutions of the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, and it is obvious to those skilled in the art that other drawings can be obtained according to these drawings without inventive effort.

FIG. 1 is a schematic flow chart of a transient stimulated Raman excitation fluorescence spectrum method provided by the invention;

FIG. 2 is a graph of excitation energy levels of a sample to be measured without degeneracy of stimulated Raman excitation pulse pairs and detection pulses in a transient stimulated Raman excitation fluorescence spectroscopy system provided by the invention;

FIG. 3 is an excitation energy level diagram of a sample to be measured under the condition that a pumping pulse and a detection pulse are degenerated in the transient stimulated Raman excitation fluorescence spectrum system provided by the invention;

FIG. 4 is a graph of excitation energy levels of a sample to be measured under the condition that a stimulated Raman excitation pulse pair and a detection pulse are degenerated in a transient stimulated Raman excitation fluorescence spectrum system provided by the invention;

FIG. 5 is a schematic diagram of a transient stimulated Raman excitation fluorescence spectrum system provided by the invention;

FIG. 6 is a schematic diagram of a second embodiment of the present invention;

FIG. 7 is a third schematic diagram of a transient stimulated Raman excitation fluorescence spectrum system provided by the invention;

FIG. 8 is a schematic diagram of a transient stimulated Raman excitation fluorescence spectrum system provided by the invention;

FIG. 9 is a schematic diagram of a transient stimulated Raman excitation fluorescence spectrum system provided by the invention;

FIG. 10 is a complete technical roadmap of the T-SREF spectroscopic technique employed by the transient stimulated Raman excitation fluorescence spectroscopy system provided by the invention;

FIG. 11 is a schematic diagram of a time domain fluorescence signal of a C=C skeleton mode generated by excitation of ATTO740 dye molecules by two pairs of stimulated Raman excitation pulses and by detection pulses in the transient stimulated Raman excitation fluorescence spectrum system provided by the invention;

fig. 12 is a spectrum diagram obtained by performing fourier transform on a time-domain fluorescence signal by a signal acquisition device in the transient stimulated raman excitation fluorescence spectrum system provided by the invention.

Detailed Description

For the purpose of making the objects, technical solutions and advantages of the present invention more apparent, the technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings, and it is apparent that the described embodiments are some embodiments of the present invention, not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.

Because the raman spectrum obtained by the existing SREF spectrum technology has a strong and inherent fluorescence background, the existing excitation technology cannot effectively inhibit the fluorescence background. Based on the above, the embodiment of the invention provides a transient stimulated raman excitation fluorescence spectrum method for generating a raman spectrum without a fluorescence background.

Fig. 1 is a schematic flow chart of a transient stimulated raman excitation fluorescence spectrum method provided in an embodiment of the present invention, as shown in fig. 1, the method includes:

s1, generating two stimulated Raman excitation pulse pairs with relative delay and adjustable relative delay based on the stimulated Raman excitation pulse pairs; the stimulated raman excitation pulse pair comprises a pump pulse and a stokes pulse which are generated based on femtosecond pulse laser and are aligned in time dimension and space dimension;

s2, based on the two stimulated Raman excitation pulse pairs, exciting a sample to be detected to generate transient stimulated Raman scattering, and based on detection pulses, exciting the sample to be detected to generate a fluorescent signal;

and S3, collecting the fluorescent signals, and carrying out Fourier transform on the fluorescent signals to obtain Raman spectra corresponding to the sample to be detected.

Specifically, the transient stimulated raman excitation fluorescence spectrum method provided by the embodiment of the invention can be realized through a transient stimulated raman excitation fluorescence spectrum system.

First, step S1 is performed to generate two stimulated raman excitation pulse pairs having a relative delay and adjustable relative delay using the stimulated raman excitation pulse pairs. The stimulated raman excitation pulse pair may be generated by a femtosecond-scale laser light source, and the stimulated raman excitation pulse pair may include a pump pulse and a stokes pulse aligned based on a time dimension and a space dimension of the femtosecond pulsed laser generation. The femtosecond laser light source may include a femtosecond laser that can be used to generate a femtosecond pulse laser.

Here, the two pairs of stimulated raman excitation pulses may be generated by a time-lapse scanning device, which may be an interferometer or the like, and is not particularly limited herein. The alignment is overlapping, the time dimension alignment means that the pump pulse and the stokes pulse are at the same position at a certain moment, that is, the pump pulse and the stokes pulse can meet each other, and the space dimension alignment means that the pump pulse and the stokes pulse are combined into the same light beam.

Then, step S2 is executed, and the sample to be measured is excited to generate transient stimulated raman scattering by using the generated two pairs of stimulated raman excitation pulses, and then is excited to generate a fluorescent signal by using the detection pulse. I.e., the sample to be tested is further excited to an electron level excited state by the detection pulse to generate a fluorescence signal (fluorescence). The fluorescence signal contains a raman resonance frequency in the time domain.

If the relative delay between the two stimulated Raman excitation pulse pairs is τ, the sample to be measured is separated in time by two timesThe vibration quantum state wave packet generated by the stimulated Raman excitation of tau and the front and back excitation is χ (t) and χ (t) respectively

Chi (t) and

differ by τ in propagation time. For each Raman mode |v in the vibrational quantum state wave packet _i >Final excitation probability P _i For coherent superposition per excitation, a phase proportional to cos (Ω _i τ) is used. Wherein Ω _i τ is the phase difference accumulated by the relative delay τ, Ω _i Is the resonance frequency of the raman mode. The quantum interference beat frequency term can be mapped to an electron energy level excited state by the detection pulse, and finally is expressed on the intensity of a read fluorescent signal.

Here, the stimulated raman excitation pulse pair and the detection pulse may be generated by one laser or may be generated by two different lasers, depending on the laser bandwidths generated by the lasers, which is not particularly limited herein.

And finally, executing step S3, collecting a fluorescence signal, and carrying out Fourier transform on the fluorescence signal to obtain a Raman spectrum corresponding to the sample to be detected. The process can be realized by a signal acquisition device, the signal acquisition device can comprise a single photon counter and a data acquisition card which are connected, the single photon counter can detect the intensity of a fluorescent signal in a time domain, and the fluorescent signal in the time domain is recorded; the time domain fluorescence signal can be subjected to Fourier transform through the data acquisition card, so that a Raman spectrum is obtained. The data acquisition card can be connected with a display through which the raman spectrum is displayed.

Here, since the fluorescence signal generated by excitation of the sample to be detected by the detection pulse includes the raman resonance frequency in the time domain, a clear vibration spectrum without fluorescence background, i.e., a raman spectrum, also referred to as a transient stimulated raman excitation fluorescence spectrum, can be seen in the frequency domain after fourier transformation. In addition, in the embodiment of the invention, the fluorescent background can be completely eliminated by windowing the time domain fluorescent signal.

Because the femtosecond pulse laser is adopted, the transient stimulated Raman excitation fluorescence spectrum method adopts an ultrafast time domain spectrum technology, namely a transient stimulated Raman excitation fluorescence (Transient Stimulated Raman Excited Fluorescence, T-SREF) spectrum technology, and the T-SREF spectrum technology excites a sample to be detected through the relative delay between two stimulated Raman excitation pulse pairs containing pumping pulses and Stokes pulses to generate a fluorescence signal containing Raman resonance frequency in the time domain, so that a clear Raman spectrum without fluorescence background can be seen in the frequency domain after Fourier transformation.

The transient stimulated Raman excitation fluorescence spectrum method provided by the invention comprises the following steps: generating two stimulated raman excitation pulse pairs with relative delays and adjustable relative delays based on the stimulated raman excitation pulse pairs; based on the two stimulated Raman excitation pulse pairs, exciting a sample to be detected to generate transient stimulated Raman scattering, and based on the detection pulse, exciting the sample to be detected to generate a fluorescent signal; and collecting a fluorescence signal, and carrying out Fourier transform on the fluorescence signal to obtain a Raman spectrum corresponding to the sample to be detected. Because two stimulated Raman excitation pulse pairs with adjustable phase-to-phase delay are introduced, a sample to be detected is excited to generate transient stimulated Raman scattering, so that the sample to be detected can generate a fluorescent signal containing Raman resonance frequency in the time domain when being excited by detection pulses, and further clear Raman spectrum without fluorescence background can be seen in the frequency domain after Fourier transformation, the quality of the Raman spectrum can be ensured, and the Raman spectrum is prevented from being influenced by the fluorescence background.

On the basis of the embodiment, the transient stimulated raman excitation fluorescence spectrum method provided by the embodiment of the invention is characterized in that the femtosecond pulse laser is generated based on a femtosecond laser;

Specifically, the degeneracy information between the stimulated raman excitation pulse pair and the detection pulse may include three cases of no degeneracy between the stimulated raman excitation pulse pair and the detection pulse, degeneracy between the pump pulse pair and the detection pulse, and degeneracy between the stimulated raman excitation pulse pair and the detection pulse, which case depends on the bandwidth of the femtosecond laser, the raman shift of the raman mode in which the sample to be measured is excited, and the energy level structure.

Wherein the stimulated raman excitation pulse pair is not degenerate to the detection pulse, and noise caused by degeneracy can be avoided. The excitation energy level diagram of the sample to be tested in this case is shown in fig. 2. Wherein τ ₂ ＝τ，τ ₁ For detecting a delay between a pulse and a pair of stimulated raman excitation pulses that is later in time than the two pairs of stimulated raman excitation pulses. I e ₀ >Is electron energy level ground state, |g>Is in a vibration ground state.

The degeneracy of the pumping pulse and the detecting pulse means that the pumping pulse and the detecting pulse are the same pulse, and the pulse covers the frequency component of the pumping pulse and the frequency component of the detecting pulse, so that the functions of the pumping pulse and the detecting pulse can be realized. The excitation energy level diagram of the sample to be tested in this case is shown in fig. 3. Wherein τ ₂ ＝τ，τ ₁ ＝0。

The stimulated raman excitation pulse pair is degenerated with the detection pulse, which means that the pumping pulse, the stokes pulse and the detection pulse are the same pulse, and the frequency component of the pumping pulse, the frequency component of the stokes pulse and the frequency component of the detection pulse are covered by the pulse, so that the functions of the pumping pulse, the stokes pulse and the detection pulse can be realized. The excitation energy level diagram of the sample to be tested in this case is shown in fig. 4.

In the embodiment of the invention, the degeneracy information between the stimulated Raman excitation pulse pair and the detection pulse can be determined according to the bandwidth of the femtosecond laser, the Raman shift of the Raman mode excited by the sample to be detected and the energy level structure, so that the structure of the femtosecond laser light source is simplified.

On the basis of the above embodiment, the method for transient stimulated raman excitation fluorescence spectroscopy provided in the embodiment of the present invention generates two stimulated raman excitation pulse pairs with a relative delay and adjustable relative delay based on the stimulated raman excitation pulse pairs, which previously includes:

Dividing the femtosecond pulse laser into a first partial beam and a second partial beam, and performing dispersion compensation on the first partial beam to obtain the Stokes pulse;

performing frequency multiplication on the second part of light beams to obtain frequency-multiplied laser, driving an optical parametric oscillator based on the frequency-multiplied laser, and outputting the pumping pulse;

the pump pulse is aligned with the stokes pulse in a time dimension as well as in a space dimension.

Specifically, when two stimulated raman excitation pulse pairs are generated, first, a femtosecond pulse laser is divided into a first partial beam and a second partial beam, and dispersion compensation is performed on the first partial beam to obtain stokes pulses. The femtosecond pulse laser can be divided into a first partial beam and a second partial beam by a polarization beam splitter aliquoting device in the femtosecond laser light source.

The first partial beam may then be dispersion compensated using a dispersion compensation device to obtain stokes pulses. The second partial beam may be frequency doubled using a frequency multiplier (SHG) to obtain a frequency doubled laser.

Thereafter, the optical parametric oscillator (Optical Parametric Oscillator, OPO) can be driven by the doubled laser to cause the OPO to output a pump pulse.

Finally, the pump pulse can be aligned with the stokes pulse in the time dimension as well as in the spatial dimension using an optical delay line.

In the embodiment of the invention, the femtosecond pulse laser is split to respectively generate the pumping pulse and the Stokes pulse, so that a light source can be provided for generating transient stimulated Raman scattering for the subsequent excitation of the sample to be detected, and the interference caused by the pumping pulse and the Stokes pulse respectively generated by different light sources is avoided.

On the basis of the above embodiment, in the transient stimulated raman excitation fluorescence spectrum method provided in the embodiment of the present invention, fourier transforming the fluorescence signal to obtain a raman spectrum corresponding to the sample to be measured includes:

Specifically, in the process of obtaining the raman spectrum corresponding to the sample to be detected, the optical background of wavelengths other than the fluorescent signal can be filtered, and fluorescent photons in the focal position in the fluorescent signal are selected, so that the signal-to-noise ratio of the raman spectrum can be improved.

And then, recording a time domain fluorescence signal corresponding to the fluorescence photon, and carrying out Fourier transform on the time domain fluorescence signal to obtain a clear Raman spectrum without fluorescence background on a frequency domain.

Based on the above embodiment, the transient stimulated raman excitation fluorescence spectrum method provided in the embodiment of the present invention, based on two pairs of stimulated raman excitation pulses, excites a sample to be measured to generate transient stimulated raman scattering, includes:

Specifically, after the stokes pulse is generated, a spectrum filtering device can be used for adjusting the bandwidth of the stokes pulse, so that the difference of the bandwidth of the stokes pulse and the bandwidth of the pump pulse is within a preset range, even if the bandwidths of the stokes pulse and the pump pulse are consistent or close, the signal to noise ratio of the fluorescent signal is improved.

Fig. 5 is a schematic structural diagram of a transient stimulated raman excitation fluorescence spectrum system provided in the embodiment of the present invention, and as shown in fig. 5, the transient stimulated raman excitation fluorescence spectrum system includes a femtosecond laser light source 1, a delay scanning device 2, and a signal acquisition device 3;

The femtosecond laser light source 1 is used for generating stimulated Raman excitation pulse pairs and detection pulses, wherein the stimulated Raman excitation pulse pairs comprise pump pulses and Stokes pulses aligned in a time dimension and a space dimension;

the time delay scanning device 2 is used for generating two stimulated raman excitation pulse pairs with relative delay and adjustable relative delay based on the stimulated raman excitation pulse pairs, wherein the two stimulated raman excitation pulse pairs are used for exciting a sample 4 to be detected to generate transient stimulated raman scattering so that the sample 4 to be detected is excited by the detection pulse to generate a fluorescent signal;

the signal acquisition device 3 is used for acquiring the fluorescence signal, and performing fourier transform on the fluorescence signal to obtain a raman spectrum corresponding to the sample 4 to be detected.

Specifically, in the transient stimulated raman excitation fluorescence spectrum system provided by the embodiment of the invention, the femtosecond laser source is used for generating a stimulated raman excitation pulse pair and a probe pulse (probe), and the stimulated raman excitation pulse pair can comprise a pump pulse (pump) and a Stokes pulse (Stokes) aligned in a time dimension and a space dimension.

The stimulated raman excitation pulse pair and the detection pulse may be generated by one laser or may be generated by two different lasers, depending on the laser bandwidth of the laser generation, and are not particularly limited herein.

The stimulated raman excitation pulse pair consisting of the pumping pulse and the stokes pulse is used for pumping molecules of a sample to be tested to a vibration excitation state positioned at the electron energy level ground state, and the pumping pulse can be a femtosecond pulse with tunable wavelength in the range of 800nm-900 nm. The wavelength range of the Stokes pulse may be 1020nm to 1050nm. The detection pulse is used for exciting molecules of the sample to be detected from a vibration excited state to an electron energy level excited state. The photon energy may be the energy of the first electron excited state of the molecule minus the vibrational excited state energy of the ground state of the electron energy level.

The delay scanning device 2 may generate two stimulated raman excitation pulse pairs with relative delay and adjustable relative delay by using the stimulated raman excitation pulse pairs, and the delay scanning device 2 may be an interferometer, where the interferometer may include two arms, a first arm and a second arm, respectively, and each arm may obtain one stimulated raman excitation pulse pair. By introducing an optical delay line in the first arm or the second arm, a relative delay between the two pairs of stimulated raman excitation pulses can be provided and can be adjusted by controlling the optical delay line.

The sample 4 to be measured may be fixed on a stage or placed in a cuvette. The sample 4 to be measured can be arranged on the transmission light paths of the two stimulated raman excitation pulse pairs and the detection pulse, the two stimulated raman excitation pulse pairs excite the sample 4 to be measured to generate transient stimulated raman scattering, and the sample to be measured is further excited to an electronic energy level excited state by the detection pulse to generate a fluorescent signal. The fluorescence signal contains a raman resonance frequency in the time domain.

In the embodiment of the invention, the relative delay tau between two stimulated Raman excitation pulse pairs, namely the delay scanning range of the delay scanning device 2, can be set to be more than 5ps, so that the resolution of the Raman spectrum determined later can be ensured to be higher than 8cm ^-1 The resolution is 1.2/5ps under a rectangular window apodization (box-car apodization) function, which is higher than the linewidth of the normal raman mode. The step length scanning of the delay scanning can be set to be smaller than 1fs, so that fluorescence intensity oscillation caused by transient absorption of the second harmonic of the laser wavelength, namely transient absorption spectra of single photons and multiple photons, can be captured.

The transient stimulated Raman excitation fluorescence spectrum system also comprises a signal acquisition device, wherein the signal acquisition device can comprise a single photon counter and a data acquisition card which are connected, and the single photon counter can be used for detecting the intensity of a fluorescence signal in a time domain and recording the fluorescence signal in the time domain; the time domain fluorescence signal can be subjected to Fourier transform through the data acquisition card, so that a Raman spectrum is obtained. The data acquisition card can be connected with a display through which the raman spectrum is displayed.

The transient stimulated Raman excitation fluorescence spectrum system provided by the embodiment of the invention comprises the following components: a femtosecond laser light source, a delay scanning device and a signal acquisition device; the femtosecond laser source is used for generating stimulated Raman excitation pulse pairs and detection pulses, wherein the stimulated Raman excitation pulse pairs comprise pump pulses and Stokes pulses aligned in a time dimension and a space dimension; the time delay scanning device is used for generating two stimulated Raman excitation pulse pairs with relative delay and adjustable relative delay based on the stimulated Raman excitation pulse pairs, and the two stimulated Raman excitation pulse pairs are used for exciting a sample to be detected to generate transient stimulated Raman scattering so that the sample to be detected is excited by the detection pulse to generate a fluorescent signal; the signal acquisition device is used for acquiring fluorescent signals and carrying out Fourier transform on the fluorescent signals to obtain Raman spectra corresponding to the sample to be detected. Because two stimulated Raman excitation pulse pairs with adjustable phase-to-phase delay are introduced, a sample to be detected is excited to generate transient stimulated Raman scattering, so that the sample to be detected can generate a fluorescent signal containing Raman resonance frequency in the time domain when being excited by detection pulses, and further clear Raman spectrum without fluorescence background can be seen in the frequency domain after Fourier transformation, the quality of the Raman spectrum can be ensured, and the Raman spectrum is prevented from being influenced by the fluorescence background.

On the basis of the above embodiment, the transient stimulated raman excitation fluorescence spectrum system provided in the embodiment of the present invention, where the femtosecond laser source includes a femtosecond laser, where the femtosecond laser is used to generate the stimulated raman excitation pulse pair; degenerate 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 to be measured, and the energy level structure.

Specifically, in the embodiment of the present invention, the femtosecond laser source includes a femtosecond laser, for example, a femtosecond fiber laser, and the femtosecond fiber laser may be a high-power ytterbium-doped femtosecond fiber laser. The femtosecond laser is used for generating stimulated raman excitation pulse pairs, and can be realized in combination with an auxiliary structure when the stimulated raman excitation pulse pairs are generated. Here, the auxiliary structure may be selected as needed, and is not particularly limited herein.

Depending on the bandwidth of the femtosecond laser, the raman shift of the excited raman mode of the sample to be measured, and the energy level structure, there may or may not be degeneracy between the stimulated raman excitation pulse pair and the detection pulse. That is, degeneracy information between the stimulated raman excitation pulse pair and the probe pulse may include three cases where there is no degeneracy between the stimulated raman excitation pulse pair and the probe pulse, where there is degeneracy between the pump pulse pair and the probe pulse pair, and where there is degeneracy between the stimulated raman excitation pulse pair and the probe pulse.

Wherein the stimulated raman excitation pulse pair is not degenerate to the detection pulse, and noise caused by degeneracy can be avoided. The femtosecond laser light source also comprises a detection laser for generating detection pulses. The excitation energy level diagram of the sample to be tested in this case is shown in fig. 2.

The pump pulse and the detection pulse are degenerated, and a laser for generating the detection pulse is not required to be introduced at this time, so that the structure of the femtosecond laser light source can be simplified, but noise caused by degeneracy is introduced. The excitation energy level diagram of the sample to be tested in this case is shown in fig. 3.

The stimulated raman excitation pulse pair is degenerate to the detection pulse, in which case the excitation energy level diagram of the sample to be measured is shown in fig. 4. In this case, only the femtosecond laser is needed, and an auxiliary structure is not needed, so that the structure of the femtosecond laser light source can be further simplified, but the noise due to degeneracy is also increased.

Furthermore, the bandwidth of the femtosecond laser is maximized in the case where the stimulated raman excitation pulse pair is degenerate with the detection pulse. The frequency components of the pumping pulse, the frequency components of the Stokes pulse and the frequency components of the detection pulse can be completely covered only when the femtosecond laser is an ultrafast femtosecond laser with ultra-large bandwidth, and the degeneracy of the stimulated Raman excitation pulse and the detection pulse can be realized.

Because the existing SREF spectrum technology uses a narrow-band picosecond laser pulse to perform single-frequency point excitation, a plurality of Raman markers cannot be excited simultaneously, and the flux is limited.

Based on the above, on the basis of the above embodiment, the transient stimulated raman excitation fluorescence spectrum system provided in the embodiment of the present invention has no degeneracy between the stimulated raman excitation pulse pair and the detection pulse, or the pump pulse and the detection pulse degeneracy; in response to this, the control unit,

the femtosecond laser light source comprises 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 for generating femtosecond pulse laser; the polarization beam splitter is used for splitting the femtosecond pulse laser into a first partial beam and a second partial beam;

Specifically, in the case where there is no degeneracy between the stimulated raman excitation pulse pair and the detection pulse, or the pump pulse and the detection pulse are degenerated, as shown in fig. 6, the femtosecond laser light sources each include a femtosecond laser 11, a polarization beam splitter 12, a first dichroic mirror 13, a first light source branch, and a second light source branch.

The parameters of the femtosecond laser 11 may include: center wavelength 1030nm, 100MHz repetition frequency, 80fs pulse width. The femtosecond laser 11 is used for generating femtosecond pulse laser.

The polarizing beam splitter 12 may split the femtosecond pulsed laser into a first partial beam having a first polarization state and a second partial beam having a second polarization state.

The first light source branch includes a first dispersion compensation device 141, and the first dispersion compensation device 141 may perform dispersion compensation on the first partial light beam, so that a pulse width of the first partial light beam approaches a fourier transform limit, and further obtain stokes pulses.

To shorten the optical path length and reduce the size of the transient stimulated raman excitation fluorescence spectroscopy system, a first mirror 142 may be introduced into the first light source branch, and the height of the first mirror 142 may be lower than the transmission optical path of the first partial light beam, so that the first partial light beam may be transmitted to the first dispersion compensating device 141 above the first mirror.

The first dispersion compensating device 141 may include two prisms and a reflecting mirror, and the first partial light beam is reflected by the reflecting mirror after passing through the prisms in sequence, and the reflected light beam is reflected by the first reflecting mirror 142 after passing through the two prisms in sequence to obtain a stokes pulse light beam. The stokes pulse may have a wavelength in the range 1020nm to 1050nm.

The second light source branch comprises a frequency multiplier 151, an optical parametric oscillator 152, a second dispersion compensation device 153 and an adjustable first optical delay line (Optical Delay Line, ODL) 154.

The frequency multiplier 151 multiplies the second partial beam, i.e., increases the frequency of the second partial beam to twice the original frequency, to obtain a frequency-multiplied laser. The optical parametric oscillator 152 is driven by the frequency-doubled laser and outputs pump pulses. The wavelength range of the pump pulse can be adjusted within the range of 800nm-900 nm.

The first optical delay line 154 may control the propagation delay of the pump pulse to align the pump pulse with the stokes pulse in the time dimension.

To shorten the optical path length and reduce the size of the transient stimulated raman excitation fluorescence spectroscopy system, a second mirror 155 may be introduced in the second light source branch, and the height of the second mirror 155 may be lower than the transmission path of the pump pulse, so that the pump pulse may be transmitted to the second dispersion compensation device 153 above the second mirror 155.

The second dispersion compensating device 153 may also include two prisms and a reflecting mirror, where the beam of the pump pulse is reflected by the reflecting mirror after passing through the prisms in sequence, and the reflected beam is reflected by the second reflecting mirror 155 after passing through the prisms in sequence to obtain a beam of the pump pulse after dispersion compensation, and the pulse width of the pump pulse is close to the fourier transform limit.

One side of the first dichroic mirror 13 receives and reflects the beam of stokes pulses, and the other side receives and transmits the beam of pump pulses, so that the pump pulses and stokes pulses are combined by wavelength difference, and the two are aligned in space dimension.

In the embodiment of the invention, the first light source branch and the second light source branch are introduced into the femtosecond laser light source, so that pumping pulses and Stokes pulses can be respectively generated, and the pumping pulses and the Stokes pulses are aligned in the time dimension and the space dimension through the first optical delay line and the first dichroic mirror, so that a light source is provided for generating transient stimulated Raman scattering for subsequently exciting a sample to be detected.

Due to the working principle of the femtosecond laser, the bandwidth of the pump pulse generated by the first light source branch and the bandwidth of the stokes pulse generated by the second light source branch are possibly inconsistent greatly, which leads to low signal-to-noise ratio of the fluorescent signal generated subsequently.

Based on the above, on the basis of the above embodiment, in the transient stimulated raman excitation fluorescence spectrum system provided in the embodiment of the present invention, the first light source branch further includes a spectrum filtering device;

Specifically, as shown in fig. 7, the first light source branch further includes a spectral filtering device 143, and the spectral filtering device 143 can adjust the bandwidth of the stokes pulse so that the difference between the bandwidths of the stokes pulse and the pump pulse is within a preset range, i.e. the bandwidths of the stokes pulse and the pump pulse are consistent or close.

Here, the spectral filtering apparatus 143 may be a 4f optical system, and may include a first grating 1431, a first lens 1432, a first slit 1433, and a mirror.

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 may be introduced in the first light source branch, the third mirror 144 may have a height lower than the transmission path of the stokes pulse, so that the stokes pulse may be transmitted over the third mirror 144 to the spectral filtering device 143.

The stokes pulse beam sequentially passes through the first grating 1431, the first lens 1432, and the first slit 1433, is reflected by a mirror, and sequentially passes through the first slit 1433, the first lens 1432, and the first grating 1431 to be output from the spectral filter device 143.

The stokes pulse beam output from the spectral filter 143 is reflected by the third mirror 144 and then further reflected by the fourth mirror 145 on one side of the first dichroic mirror 13.

On the basis of the embodiment, the transient stimulated raman excitation fluorescence spectrum system provided by the embodiment of the invention has no degeneracy between the stimulated raman excitation pulse pair and the detection pulse; in response to this, the control unit,

the detection laser is used for generating the detection pulse;

Specifically, as shown in fig. 8, in the case where there is no degeneracy between the stimulated raman excitation pulse pair and the detection pulse, the femtosecond-level laser light source further includes a detection laser 16, and a second optical delay line 17 and a second dichroic mirror 18 disposed along the optical path.

The type of the detection laser used for generating the detection pulse may be selected as required, and is not particularly limited herein, as long as the detection pulse can be generated.

The second optical delay line 17 can control the delay tau existing between the probe pulse and the target pulse pair ₁ I.e. to control the delay existing between the probe pulse and the stimulated raman excitation pulse pair that is later in time than the two stimulated raman excitation pulse pairs. The delay τ ₁ The range of the value of (2) can be 0-5ps for detecting the life of the electronic energy level of the sample to be detected. Delay τ ₁ The greater the value of (2), the weaker the intensity of the fluorescent signal, and the delay τ ₁ When the value of (2) is 0, the intensity of the fluorescent signal is the strongest.

The second dichroic mirror 18 is used to align the two stimulated raman excitation pulse pairs with the detection pulse in the spatial dimension, i.e. to combine the beams using wavelength differences.

Based on the above embodiments, the transient stimulated raman excitation fluorescence spectrum system provided in the embodiments of the present invention, the delay scanning device includes an interferometer, and an arm of the interferometer includes an adjustable third optical delay line.

Specifically, the delay scanning device may be implemented by an interferometer, which may be a mach-zehnder interferometer, a michelson interferometer, or the like, and is not limited herein. The interferometer may comprise two arms for outputting a pair of stimulated raman excitation pulses, respectively. One of the two arms of the interferometer includes an adjustable third optical delay line for controlling the magnitude of the relative delay between the two pairs of stimulated raman excitation pulses output by the interferometer.

On the basis of the embodiment, the transient stimulated raman excitation fluorescence spectrum system provided by the embodiment of the invention is characterized in that the third optical delay line is fixed on the displacement table;

Specifically, the third optical delay line can be fixed on the displacement table, and the movement of the displacement table drives the movement of the third optical delay line, so that the control of the relative delay between the two stimulated raman excitation pulse pairs is realized.

The signal acquisition device can be connected with the delay scanning device, and particularly can be connected with a displacement table in the delay scanning device, so that the signal acquisition device is triggered to acquire fluorescent signals and carry out Fourier transform on the fluorescent signals when the position information of the displacement table changes, and thus the synchronous acquisition of the relative delay of two stimulated Raman excitation pulse pairs and the intensity of the fluorescent signals can be realized, and the accuracy and precision of the signals and the Raman spectrum are improved.

Since the translation stage movement is not perfectly uniform, this will lead to deviations in the control of the relative delays, which in turn affect the accurate acquisition of the fluorescence signal and the accuracy of the raman spectrum. Based on the above, on the basis of the above embodiment, the transient stimulated raman excitation fluorescence spectrum system provided in the embodiment of the present invention further includes a first beam splitter, a second beam splitter, and a reference signal detection device;

Specifically, as shown in fig. 9, the interferometer further includes a first beam splitter 21, a second beam splitter 22, and a reference signal detection device. The first beam splitter 21 and the second beam splitter 22 may be both half-transmissive and half-reflective beam splitters, and the transmittance may be about 50%.

The first beam splitter 21 is configured to split a target beam of the stimulated raman excitation pulse pair generated by the femto-second laser light source into a third partial beam and a fourth partial beam, where the third partial beam is transmitted along a first arm of the interferometer, and the fourth partial beam is transmitted along a second arm of the interferometer. In fig. 8, the first arm of the interferometer has an adjustable third optical delay line 23 and the second arm of the interferometer has a fixed fourth optical delay line 24.

The second beam splitter 22 is used for combining the light beam output by the first arm and the light beam output by the second arm, and obtaining a fifth part of light beam and a sixth part of light beam respectively. The fifth light beam may be incident on the sample 4 to be measured via a mirror.

In fig. 9, in order to shorten the optical path length and reduce the size of the transient stimulated raman excitation fluorescence spectrum system, a third dichroic mirror 5 and a fifth reflecting mirror 6 are introduced into the transient stimulated raman excitation fluorescence spectrum system, and a fifth part of light beams are reflected by the third dichroic mirror 5, reflected by the fifth reflecting mirror 6, and then reflected by one reflecting mirror to an objective lens 7 after being expanded by two lenses, so that two stimulated raman excitation pulse pairs and detection pulses are incident on a sample 4 to be measured on a stage through the objective lens 7 and excite the sample 4 to be measured.

After that, the fluorescent signal generated by the excitation of the sample 4 to be measured by the detection pulse is also received by the objective lens 7 and is collected by the signal collection device 3 through the third dichroic mirror 5.

The reference signal detection means may receive the sixth portion of the light beams and may convert the sixth portion of the light beams into reference signals having a coherence time greater than a preset threshold. The preset threshold value may be set as needed here, and may be set to 10ps, for example. The reference signal is narrowband high coherence light. Calibration of the position information of the translation stage can be achieved by interference fringes of the reference signal.

According to the embodiment of the invention, the reference signal detection device is introduced, so that the situation that deviation occurs in control of relative delay caused by non-uniform movement of the translation stage can be avoided, the accurate collection of fluorescent signals can be realized, and the accuracy of Raman spectrum can be further improved.

On the basis of the above embodiment, the transient stimulated raman excitation fluorescence spectrum system provided in the embodiment of the present invention, the reference signal detection device may include a grating, a lens, a slit, and a photodiode;

the sixth part of light beams sequentially pass through the grating, the lens and the slit to obtain the reference signal;

The photodiode is used for detecting the reference signal.

Specifically, as shown in fig. 9, in order to distinguish the grating, the lens, and the slit in the reference signal detecting apparatus from the spectral filtering apparatus 143, the grating in the reference signal detecting apparatus is denoted as a second grating 231, the lens is denoted as a second lens 232, and the slit is denoted as a second slit 233.

The sixth part of the light beams sequentially passes through the second light beam 231, the second lens 232 and the second slit 233 to realize spectral filtering, so as to obtain a reference signal. The reference signal may be detected by a Photodiode (PD) 234.

On the basis of the embodiment, the signal acquisition device comprises an optical filter, a screening element, a photoelectric detector and a data acquisition card, wherein the optical filter, the screening element, the photoelectric detector and the data acquisition card are arranged along an optical path;

In particular, the screening element may comprise confocal apertures and/or multimode optical fibers and the photodetector may comprise a single photon counter or other photodetector. As shown in fig. 9, the signal acquisition device 3 may include a filter 31, a screening element 32, a photodetector 33, and a data acquisition card 34 disposed along the optical path. The filter 31 may be used to filter out noise signals in the fluorescence signal, and the screening element 32 may be used to select out fluorescence photons in the fluorescence signal that are in the focal position. In the embodiment of the invention, the signal to noise ratio of the Raman spectrum can be improved due to the introduction of the optical filter and the screening element.

The photodetector 33 may have high quantum efficiency, and be used to record a time domain fluorescence signal corresponding to the fluorescence photon, and the data acquisition card 34 may be used to perform fourier transform on the time domain fluorescence signal, so as to obtain a raman spectrum.

In summary, the transient stimulated raman excitation fluorescence spectrum system provided by the embodiment of the invention can realize the detection sensitivity of the raman mode of the sample to be detected not lower than 40nM by the T-SREF spectrum technology. The T-SREF spectroscopy technique can acquire Raman spectra and observe a large number of transient spectra of multiphoton absorption. Compared with the traditional SREF spectrum technology, the transient stimulated Raman excitation fluorescence spectrum system can eliminate the inherent fluorescence background and realize the hyperspectral data acquisition of which the single excitation covers hundreds of wavenumbers on the premise of having the same sensitivity, and the main technical bottleneck faced by the traditional SREF spectrum technology is solved at one time.

The T-SREF spectrum technology can be used in various fields such as hyperchromatic multiplexing fluorescence microscopy imaging, space transcriptome imaging, space protein group imaging, transient Raman spectrum imaging, electric field sensing imaging and the like. The T-SREF spectrum technology is used for simultaneously acquiring Raman spectrum and transient absorption spectrum of single photon and multiple photons.

FIG. 10 is a complete technical roadmap of the T-SREF spectroscopic technique, as shown in FIG. 10, with no degeneracy between the stimulated Raman excitation pulse pairs and the probe pulses, and with the pump pulses and Stokes pulses aligned in both the time and space dimensions, two stimulated Raman excitation pulse pairs having adjustable phase-to-phase delays are generated by a time-lapse scanning device. On one hand, the relative delay generated by the delay scanning device is triggered synchronously with the acquisition action of the signal acquisition device; on the other hand, the detection pulse has a delay in the time dimension with respect to the stimulated raman excitation pulse pair that is temporally later than the two stimulated raman excitation pulse pairs generated by the delay scanning device, but is aligned in the spatial dimension. And finally, exciting the sample to be detected by using a light beam containing a pumping pulse, a Stokes pulse and a detection pulse, collecting a fluorescent signal generated by the sample to be detected by using a signal collecting device, and carrying out Fourier transform on the fluorescent signal to finally obtain a Raman spectrum.

Taking the sample to be tested as ATTO740 dye as an example, the ATTO740 dye molecule is excited by two stimulated raman excitation pulse pairs to generate transient stimulated raman scattering, and is excited by the detection pulse to generate a time domain fluorescent signal in a c=c framework mode, as shown in fig. 11. In fig. 11, a rectangular window width of 4.5ps is taken as an example, and the rectangular window width is the relative delay τ between two stimulated raman excitation pulse pairs.

Fig. 12 is a spectrum diagram obtained by fourier transforming the time domain fluorescent signal in fig. 11 by the signal acquisition device. In FIG. 12, 1642cm ^-1 The spectral line at the position is the Raman spectrum of the C=C framework mode of the ATTO740 dye molecule, divided by 1642cm ^-1 The other spectral lines are captured electron energy level transient absorption spectra of single photons and multiphoton.

The apparatus embodiments described above are merely illustrative, wherein the elements illustrated as separate elements may or may not be physically separate, and the elements shown as elements may or may not be physical elements, may be located in one place, or may be distributed over a plurality of network elements. Some or all of the modules may be selected according to actual needs to achieve the purpose of the solution of this embodiment. Those of ordinary skill in the art will understand and implement the present invention without undue burden.

From the above description of the embodiments, it will be apparent to those skilled in the art that the embodiments may be implemented by means of software plus necessary general hardware platforms, or of course may be implemented by means of hardware. Based on this understanding, the foregoing technical solution may be embodied essentially or in a part contributing to the prior art in the form of a software product, which may be stored in a computer readable storage medium, such as ROM/RAM, a magnetic disk, an optical disk, etc., including several instructions for causing a computer device (which may be a personal computer, a server, or a network device, etc.) to execute the method described in the respective embodiments or some parts of the embodiments.

Finally, it should be noted that: the above embodiments are only for illustrating the technical solution of the present invention, and are not limiting; although the invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical scheme described in the foregoing embodiments can be modified or some technical features thereof can be replaced by equivalents; such modifications and substitutions do not depart 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, comprising:

2. The transient stimulated raman excitation fluorescence spectroscopy method of claim 1, wherein the femtosecond pulsed laser is generated based on a femtosecond laser;

3. The method of transient stimulated raman excitation fluorescence spectroscopy of claim 1, wherein fourier transforming the fluorescence signal to obtain a raman spectrum corresponding to the sample to be measured comprises:

4. A method according to any one of claims 1-3, wherein the excitation of the sample to be measured to produce transient stimulated raman scattering based on two pairs of said stimulated raman excitation pulses is preceded by:

5. A transient stimulated raman excitation fluorescence spectroscopy system, comprising: a femtosecond laser light source, a delay scanning device and a signal acquisition device;

6. The transient stimulated raman excitation fluorescence spectroscopy system of claim 5, wherein said femtosecond laser light source comprises a femtosecond laser for generating said femtosecond pulsed laser;

7. The transient stimulated raman excitation fluorescence spectroscopy system of claim 5, wherein there is no degeneracy between the stimulated raman excitation pulse pair and the probe pulse, or the pump pulse is degenerated with the probe pulse; in response to this, the control unit,

8. The transient stimulated raman excitation fluorescence spectroscopy system of claim 7, wherein said first light source branch further comprises a spectral filtering device;

9. The transient stimulated raman excitation fluorescence spectroscopy system of claim 7, wherein there is no degeneracy between the stimulated raman excitation pulse pair and the probe pulse; in response to this, the control unit,

the detection laser is used for generating the detection pulse;

10. The transient stimulated raman excitation fluorescence spectroscopy system of any one of claims 5-9, wherein the time-lapse scanning device comprises an interferometer, an arm of which comprises an adjustable third optical delay line.

11. The transient stimulated raman excitation fluorescence spectroscopy system of claim 10, wherein the third optical delay line is fixed on a displacement stage;

12. The transient stimulated raman excitation fluorescence spectroscopy system of claim 11, wherein the interferometer further comprises a first beam splitter, a second beam splitter, and a reference signal detection device;

13. The transient stimulated raman excitation fluorescence spectroscopy system of claim 12, wherein the reference signal detection means comprises a grating, a lens, a slit, and a photodiode;

the photodiode is used for detecting the reference signal.

14. The transient stimulated raman excitation fluorescence spectroscopy system of any one of claims 5-9, wherein the signal acquisition device comprises an optical filter, a screening element, a photodetector, and a data acquisition card disposed along an optical path;