Tunable excited Raman spectrum microscopic imaging method and device

Abstract

The invention discloses a tunable excited Raman spectrum microimaging method and device, and belongs to the field of spectrum detection and optical imaging. The device comprises a broadband adjustable wavelength light source, a high-resolution wavelength scanning module, a stimulated Raman signal excitation module and a stimulated Raman signal detection module. The stimulated Raman signal excitation and imaging module is used for focusing the two beams of narrow-band femtosecond pulse light which are overlapped in time and space on a sample and exciting a stimulated Raman signal, and meanwhile, moving the sample to realize spot scanning microscopic imaging; and the stimulated Raman signal detection module is used for converting the stimulated Raman scattering signal into an electric signal, demodulating the electric signal by using a lock-in amplifier, and recording spectral information by using a data acquisition card. According to the invention, the broadband tunable characteristic of the optical parametric amplifier is utilized to tune the pump light to the wavelength region of the sample absorption peak length so as to realize electron pre-resonance stimulated Raman; and a balance detection technology is combined at a detection end, so that high-sensitivity, high-resolution and high-signal-to-noise-ratio detection of stimulated Raman signals is realized.

Description

Technical Field

[0001] The invention relates to a tunable excitation stimulated Raman spectroscopy microscopic imaging method and device, belonging to the technical field of spectral detection and optical imaging. Background Art

[0002] Stimulated Raman spectroscopy (SRS) is a third-order nonlinear optical process used to study the molecular vibration characteristics of matter. In SRS, the interaction between two photons and a molecule is involved. When a pump photon and a Stokes photon are irradiated on a molecule at the same time, if the energy difference between the two photons is equal to the vibration energy level difference of the molecule, the molecule will be excited to a virtual energy level and quickly jump down to its vibration energy level. This will lead to the enhancement of Stokes photons and the attenuation of pump photons. Therefore, the SRS signal can be obtained by detecting the intensity change of pump photons or Stokes photons.

[0003] Although stimulated Raman spectroscopy detection technology has become increasingly mature, the stimulated Raman scattering process is usually extremely weak, especially in low-concentration samples. In order to obtain a sufficiently strong signal, it may be necessary to increase the laser power or scan multiple times, further increasing the time and complexity of the experiment. In addition, the detection sensitivity of Raman scattering is low and it is easily interfered by the fluorescence background. On the other hand, most of the pump light sources used in existing technologies are ultrafast lasers based on optical parametric oscillators, while Stokes light is the direct output of the laser, which greatly limits the practical application of stimulated Raman scattering technology. In order to solve the problems of narrow excitation wavelength range, few types of measurable samples, and low signal-to-noise ratio of existing methods, and to achieve efficient excitation of Raman vibration states, so as to expand stimulated Raman spectroscopy detection technology to a wider range of applications, further research is still needed. Summary of the invention

[0004] In order to solve the problems of low signal-to-noise ratio, limited wavelength adjustment range and insufficient detection sensitivity of stimulated Raman spectroscopy detection systems in the prior art, the purpose of the present invention is to provide a tunable excited stimulated Raman spectroscopy microscopic imaging method and device, which utilizes the wide-band tunable characteristics of an optical parametric amplifier to tune the excitation wavelength to the electron pre-resonance range, and combines balanced detection technology at the detection end to achieve high-sensitivity, high-resolution, and high-signal-to-noise ratio detection of stimulated Raman scattering signals.

[0005] The objective of the present invention is achieved through the following technical solutions:

[0006] The present invention discloses a tunable stimulated Raman spectroscopy microscopic imaging method, comprising the following steps:

[0007] Step 1: Select the sample to be tested and determine the energy level structure and Raman wave number of the Raman mode to be measured; the Raman wave number is given by the formula Calculate, where Ω represents the Raman wave number, λp represents the pump light wavelength, and λs represents the Stokes light wavelength;

[0008] Step 2: The laser light emitted by the femtosecond laser is divided into two beams by the first beam splitter, one beam enters the stimulated Raman pump pulse light source in the tunable broadband femtosecond pulse laser light source, and the other beam is reflected by the first reflector and enters the stimulated Raman Stokes pulse light source in the tunable broadband femtosecond pulse laser light source; according to the energy level structure and Raman wave number selected in step 1, the central wavelengths of the output lights of the broadband femtosecond pump pulse laser light source and the broadband femtosecond Stokes pulse laser light source are adjusted to achieve precise wavelength adjustment of the pump light and the Stokes light to match the Raman vibration mode of the sample; at the same time, the wavelength of the pump excitation light is tuned to the region where the absorption peak of the sample molecule is slightly offset to a long wavelength to achieve an electron pre-resonance effect; this effect greatly improves the intensity of the stimulated Raman signal by enhancing the electron-vibration coupling mode of the target molecule, while reducing the interference of background signals such as fluorescence;

[0009] Step 3, collimate the laser output by the broadband femtosecond pulse laser light source in step 2 and input it into a high-resolution wavelength scanning monochromator, adjust the slit width and the rotation angle of the diffraction grating to output a narrow-band femtosecond pulse laser; wherein the first high-resolution wavelength scanning monochromator receives the output light of the broadband femtosecond pump pulse laser light source, and controls the rotation of its grating by an electric rotating stage to achieve continuous wavelength scanning; the output narrow-band pump laser is then transmitted to the time delay line in the stimulated Raman signal excitation module; the output light of the broadband femtosecond Stokes pulse laser light source is received by the second high-resolution wavelength scanning monochromator, and a narrow-band Stokes laser is output, which is then transmitted to the second beam reduction system in the stimulated Raman signal excitation module;

[0010] Step 4: The narrow-band pump pulse light output by the first high-resolution wavelength scanning monochromator enters the stimulated Raman signal excitation module, passes through the time delay line, the first beam reduction system, and the second beam splitter in sequence, and is reflected by the dichroic mirror; at the same time, the narrow-band Stokes pulse light output by the second high-resolution wavelength scanning monochromator enters the stimulated Raman signal excitation module, passes through the second beam reduction system, the 1 / 4 wave plate, the optical modulator, the Glan Taylor prism, and the second reflector in sequence, and is transmitted by the dichroic mirror; the two narrow-band pulse lasers combined by the dichroic mirror are used to focus by the microscope objective lens The stimulated Raman signal is excited on the sample placed on the sample stage; the signal is converged by the second converging mirror and filtered out by the filter and the second continuous neutral density filter to remove the background light and then collected by the second photodetector as the signal light; before the two narrow-band pulse laser beams are combined by the dichroic mirror, the pump light is split by the second beam splitter, and the reflected pump light passes through the first converging mirror and the first continuous neutral density filter and is collected by the first photodetector as the reference light; during imaging, the sample is moved on the XYZ three-dimensional sample stage controlled by the program to realize point scanning;

[0011] Step 5: Input the voltage signals collected by the first photodetector and the second photodetector in step 4 into a lock-in amplifier for differential amplification and demodulation to obtain the stimulated Raman signal; the stimulated Raman signal is collected by a data acquisition card controlled by Labview software to achieve stimulated Raman spectrum measurement and imaging of the sample selected in step 1;

[0012] Step 6: Tuning the central wavelengths of the output lights of the broadband femtosecond pump pulse laser light source (401) and the broadband femtosecond Stokes pulse laser light source (402), repeating steps 1 to 6 to achieve different Raman mode measurements, that is, achieving tunable excitation stimulated Raman spectroscopy microscopy imaging.

[0013] Furthermore, the time delay line is arranged between the first high-resolution wavelength scanning monochromator and the first beam reduction system in the pump pulse optical path, and is used to change the optical path difference between the pump light and the Stokes light, so that the pump pulse and the Stokes pulse are aligned in the time dimension.

[0014] Furthermore, the first beam reduction system and the second beam reduction system are used to spatially reduce the two pulsed light beams to couple the light modulator with the microscope objective aperture.

[0015] Furthermore, the quarter wave plate is used to convert the Stokes light from linear polarization to elliptically polarized light.

[0016] Furthermore, the Glan Taylor prism is used to change the modulated light from elliptically polarized light to linear polarized light.

[0017] Furthermore, the signal generator outputs a modulation signal, which is amplified by a voltage amplifier and then sent to an optical modulator for modulating the intensity of the pulsed light.

[0018] Furthermore, the light intensity modulation system composed of the quarter wave plate, the light modulator and the Glan-Taylor prism is placed between the beam reduction system of the pump light path or the Stokes light path and the dichroic mirror.

[0019] Furthermore, the dichroic mirror is used to spatially combine the Stokes light and the narrow-band pump light so that the pump pulse and the Stokes pulse are aligned in the spatial dimension.

[0020] Furthermore, the signal generator and the data acquisition card are synchronously triggered by a laser; the signal generator inputs a signal with the same frequency as the modulation signal into the phase-locked amplifier and is synchronously triggered.

[0021] Furthermore, before measuring the stimulated Raman signal, the Stokes light source is first blocked, the first continuous neutral density filter and the second continuous neutral density filter are adjusted, and the photoelectric signals received by the first photodetector and the second photodetector are balanced so that the voltage signal obtained after the differential operation of the phase-locked amplifier is close to 0, thereby forming a balanced detection structure to eliminate optical path noise and improve the signal-to-noise ratio.

[0022] The tunable excitation stimulated Raman spectroscopy microscopy imaging device disclosed in the present invention is used to implement a tunable excitation stimulated Raman spectroscopy microscopy imaging method. The tunable excitation stimulated Raman spectroscopy microscopy imaging device comprises a wide-band tunable wavelength laser light source module, a high-resolution wavelength scanning module, a stimulated Raman signal excitation module and a stimulated Raman signal detection module.

[0023] The wide-band adjustable wavelength laser light source module based on an optical parametric amplifier is used to output two beams of broadband femtosecond pulse lasers with different wavelengths and continuously adjustable wavelengths; it consists of a femtosecond laser, a first beam splitter, a first reflector and a tunable broadband femtosecond pulse laser light source.

[0024] The high-resolution wavelength scanning module is used to achieve spectral dispersion of the output lasers of the broadband femtosecond pump pulse laser light source and the broadband femtosecond Stokes pulse laser light source, and output two narrow-band femtosecond pulse lights; it is composed of a first high-resolution wavelength scanning monochromator and a second high-resolution wavelength scanning monochromator.

[0025] The stimulated Raman scattering signal excitation module is used to focus two narrow-band femtosecond pulse lights that overlap in time and space on the sample and excite the stimulated Raman signal; it consists of a time delay line, a first beam reduction system, a second beam reduction system, a 1 / 4 wave plate, an optical modulator, a Glan-Taylor prism, a second reflector, a second beam splitter, a dichroic mirror, a microscope objective, a sample stage, a signal generator, and a voltage amplifier.

[0026] The stimulated Raman signal detection module is used to collect the stimulated Raman scattering signal and convert it into an electrical signal, and use a phase-locked amplifier to demodulate the signal and then perform data acquisition; it is composed of a second beam splitter, a first converging mirror, a first continuous neutral density filter, a first photodetector, a second converging mirror, a shortwave pass filter, a second continuous neutral density filter, a second photodetector, a signal generator, a phase-locked amplifier and a data acquisition card.

[0027] Furthermore, the first high-resolution wavelength scanning monochromator and the second high-resolution wavelength scanning monochromator have the same structure, and a 4f system is formed by a third reflector, a plane diffraction blazed grating, a cylindrical mirror, an adjustable slit and a roof mirror; the broadband pulse lasers output by the stimulated Raman pump pulse light source and the stimulated Raman-Stokes pulse light source enter the first high-resolution wavelength scanning monochromator and the second high-resolution wavelength scanning monochromator respectively, and are reflected by the third reflector to the plane diffraction blazed grating for dispersion and spectroscopy, wherein the first-order diffraction light is converged by the cylindrical mirror and outputs a narrow-band femtosecond pulse light through the adjustable slit, and the pulse light is reflected back by the roof mirror and returns along the original optical path, thereby ensuring that the optical path structure is not changed during the wavelength scanning.

[0028] Furthermore, the optical modulator is an electro-optic modulator or an acousto-optic modulator for intensity modulation of the laser.

[0029] Furthermore, if the light intensity modulation system composed of a quarter wave plate, an optical modulator and a Glan-Taylor prism is placed between the first beam reduction system and the dichroic mirror in the pump light path, the stimulated Raman gain signal is detected, and at this time the filter is a long-wave pass filter used to filter out excess pump light noise;

[0030] If the light intensity modulation system composed of a 1 / 4 wave plate, an optical modulator and a Glan-Taylor prism is placed between the second beam reduction system and the dichroic mirror in the Stokes optical path, the stimulated Raman loss signal is detected. At this time, the filter is a short-wave pass filter used to filter out excess Stokes light noise.

[0031] Beneficial effects:

[0032] 1. The tunable excited stimulated Raman spectroscopy microscopy imaging method and device disclosed in the present invention utilizes the wide-band tunable characteristics of an optical parametric amplifier to construct two tunable laser light sources, which can generate a wide-band, tunable femtosecond pulse light source. Compared with the common solution of using an optical parametric amplifier in combination with a fixed wavelength light source, it can cover a wider wavelength range and is suitable for spectral detection of different Raman modes of various substances.

[0033] 2. The tunable excitation stimulated Raman spectroscopy microscopy imaging method and device disclosed in the present invention adopts balanced detection technology at the detection end, which can simultaneously detect signal light and reference light. Compared with traditional detection technology, this method can better suppress laser noise, environmental interference and background signal interference in the detection process, and enhance the system's weak signal detection capability.

[0034] 3. The tunable excitation stimulated Raman spectroscopy microscopy imaging method and device disclosed in the present invention tunes the wavelength of the pump excitation light to a region where the absorption peak of the sample molecule is slightly shifted toward a longer wavelength to achieve an electron pre-resonance effect; this effect can greatly increase the intensity of the stimulated Raman signal by enhancing the electron-vibration coupling mode of the target molecule, while reducing the interference of the background signal.

[0035] 4. The present invention discloses a tunable excited stimulated Raman spectroscopy microscopy imaging method and device, wherein a high-resolution wavelength scanning monochromator is composed of a third reflector, a plane diffraction blazed grating, a cylindrical mirror, an adjustable slit and a roof mirror to form a 4f system; the first-order diffraction light after dispersion and spectroscopy on the plane diffraction grating is converged by the cylindrical mirror and outputted through the adjustable slit as a narrow-band femtosecond pulse light, which is reflected back by the roof mirror and returns along the original optical path to ensure that the optical path structure is not changed during the wavelength scanning. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1 is a schematic diagram of the logical structure of a tunable excitation stimulated Raman spectroscopy microscopy imaging method according to an embodiment of the present invention;

[0037] Figure 2 is a diagram of the optical path structure of a tunable excitation stimulated Raman spectroscopy microscopy imaging device according to an embodiment of the present invention;

[0038] Among them: 1-femtosecond laser, 2-first beam splitter, 3-first reflector, 4-wideband tunable laser light source, 401-wideband tunable wavelength pump light source, 402-wideband tunable wavelength Stokes light source, 5-high-resolution wavelength scanning monochromator, 501-first high-resolution wavelength scanning monochromator, 502-second high-resolution wavelength scanning monochromator, 6-stimulated Raman scattering signal excitation module, 601-time delay line, 602-first beam reduction system, 603-second beam reduction system, 604-quarter wave plate, 605 -electro-optic modulator, 606-Glan-Taylor prism, 607-second reflector, 608-second beam splitter, 609-dichroic mirror, 610-microscope objective, 611-sample stage, 612-signal generator, 613-voltage amplifier, 701-first converging mirror, 702-first continuous neutral density filter, 703-first photodetector, 704-second converging mirror, 705-low-pass filter, 706-second continuous neutral density filter, 707-second photodetector, 708-phase-locked amplifier, 709-data acquisition card.

[0039] Figure 3 is a diagram showing the optical path structure of a high-resolution wavelength scanning monochromator according to an embodiment of the present invention;

[0040] Among them: 8-the third reflecting mirror, 9-the plane diffraction blazed grating, 10-the cylindrical mirror, 11-the adjustable slit, 12-the roof mirror. DETAILED DESCRIPTION

[0041] In order to better illustrate the purpose and advantages of the present invention, the invention is further described below with reference to the accompanying drawings and examples.

[0042] The present invention utilizes the wide-band adjustable characteristics of the optical parametric amplifier, combines the electronic pre-resonance effect with the balanced detection technology, and can realize the detection of stimulated Raman signals with high sensitivity, high resolution, and high signal-to-noise ratio.

[0043] Embodiment 1:

[0044] like Figure 1 As shown, a schematic diagram of the logical structure of the tunable excitation stimulated Raman spectroscopy microscopy imaging method provided by an embodiment of the present invention is shown, in which a broadband laser is generated by a wide-band tunable femtosecond pump light source and a wide-band tunable femtosecond Stokes light source, and then a narrow-band laser is generated by dispersion spectroscopy through a high-resolution wavelength scanning monochromator; two narrow-band laser beams that overlap in time and space are focused at the sample to excite electronic pre-resonance stimulated Raman signals, and the sample is moved to realize point scanning tunable excitation stimulated Raman spectroscopy microscopy imaging.

[0045] like Figure 2 As shown, an embodiment diagram of a tunable stimulated Raman spectroscopy microscopy imaging device provided by an embodiment of the present invention. The device described in this example measures a stimulated Raman loss signal, including a femtosecond laser 1, a first beam splitter 2, a first reflector 3, a wide-band tunable laser light source 4, a wide-band tunable wavelength pump light source 401, a wide-band tunable wavelength Stokes light source 402, a high-resolution wavelength scanning monochromator 5, a first high-resolution wavelength scanning monochromator 501, a second high-resolution wavelength scanning monochromator 502, a stimulated Raman scattering signal excitation module 6, a time delay line 601, a first beam reduction system 602, a second beam reduction system 603, a quad splitter A wave plate 604, an electro-optic modulator 605, a Glan Taylor prism 606, a second reflector 607, a second beam splitter 608, a dichroic mirror 609, a microscope objective 610, a sample stage 611, a signal generator 612, a voltage amplifier 613, a first converging lens 701, a first continuous neutral density filter 702, a first photodetector 703, a second converging lens 704, a shortwave pass filter 705, a second continuous neutral density filter 706, a second photodetector 707, a phase-locked amplifier 708, and a data acquisition card 709.

[0046] like Figure 3As shown, the optical path structure diagram of the high-resolution wavelength scanning monochromator provided in an embodiment of the present invention; the two high-resolution wavelength scanning monochromators have the same structure, and the 4f system is composed of a third reflector 8, a plane diffraction blazed grating 9, a cylindrical mirror 10, an adjustable slit 11, and a roof mirror 12.

[0047] The broadband pulse lasers output by the stimulated Raman pump pulse light source 401 and the stimulated Raman Stokes pulse light source 402 enter the first high-resolution wavelength scanning monochromator 501 and the second high-resolution wavelength scanning monochromator 502 respectively, and are then reflected by the third reflector 8 to the plane diffraction blazed grating 9 controlled by the electric rotating table for dispersion and spectroscopy. The first-order diffraction light is converged by the cylindrical mirror 10 and output as a narrow-band femtosecond pulse laser through the adjustable slit 11; the narrow-band femtosecond pulse light is reflected by the roof mirror 12 and returns along the original optical path, ensuring that the optical path structure is not changed during the wavelength scanning.

[0048] The specific parameters of each device described in this embodiment are as follows:

[0049] Femtosecond laser 1 is a 1030 nm infrared laser with a pulse width of 290 fs, a repetition rate of 100 kHz, and an output energy of 200 μJ;

[0050] The wide-band tunable laser light source 4 generates a wide-band tunable wavelength laser light source that can cover a wide wavelength range from visible to near infrared (600nm-950nm), has a half-width of 8nm, and a spot diameter of 4mm, so that the system can detect Raman vibration modes of multiple molecules;

[0051] The grating 9 is a plane diffraction blazed grating with a line count of 1200 lines / mm and a central wavelength of 750nm; the focal length of the cylindrical mirror 10 is 250mm; the first high-resolution wavelength scanning monochromator 501 and the second high-resolution wavelength scanning monochromator 502 have a wavelength resolution of 0.2nm to achieve fine wavelength scanning;

[0052] The first beam reduction system 602 and the second beam reduction system 603 have a beam reduction ratio of 2:1, and are used to reduce the Stokes spot size to 2 mm so as to be coupled into the light modulator 605 and the microscope objective 610;

[0053] The optical modulator 605 is an electro-optical modulator with a light inlet diameter of 2 mm. In this example, it is placed between the second beam reduction system 603 and the second reflector 607 in the Stokes optical path to modulate the intensity of the Stokes light.

[0054] The signal generator 612 outputs a square wave signal with a frequency of 5kHz and a high and low level of ±8V to the electro-optical modulator 605 to modulate the Stokes light intensity, and its modulation efficiency is greater than 95%;

[0055] The filter 705 adopts a short-wave pass filter with a cut-off wavelength of 850nm;

[0056] The tunable excitation stimulated Raman spectroscopy microscopy imaging method disclosed in this embodiment is specifically implemented in the following steps:

[0057] Step 1: Select the sample to be tested and determine the energy level structure and Raman wave number to be measured; the Raman wave number is calculated by the formula Calculate, where Ω represents the Raman wave number, λp represents the pump light wavelength, and λs represents the Stokes light wavelength; taking the detection of the C≡N bond of the Rhodamine-800 sample molecule as an example, the required Raman wave number is 2236cm -1 ;

[0058] Step 2: The laser light emitted by the femtosecond laser 1 is divided into two beams by the first beam splitter 2, one beam enters the stimulated Raman pump pulse light source 401 in the tunable broadband femtosecond pulse laser light source 4, and the other beam is reflected by the first reflector 3 and enters the stimulated Raman Stokes pulse light source 402 in the tunable broadband femtosecond pulse laser light source 4; according to the energy level structure and Raman wave number selected in step 1, the central wavelengths of the output lights of the broadband femtosecond pump pulse laser light source 401 and the broadband femtosecond Stokes pulse laser light source 402 are adjusted to achieve precise wavelength adjustment of the pump light and the Stokes light to match the specific Raman vibration mode of the sample; at the same time, the wavelength of the pump excitation light is tuned to the region where the absorption peak of the target sample molecule is slightly offset to a long wavelength to achieve an electron pre-resonance effect; this effect greatly improves the intensity of the stimulated Raman signal by enhancing the electron-vibration coupling mode of the target molecule, while reducing the interference of the background signal;

[0059] The electron pre-resonance effect process is realized according to the following formula:

[0060]

[0061] Where σ is the electron pre-resonance Raman cross section; ω0 is the electron absorption center frequency of the molecule, that is, the resonance frequency; ω pump is the frequency of the excitation light (pump light); Γ e is the line width of the molecular electronic state (width of the absorption band); K is a constant related to the molecular properties; ω vib is the frequency of the vibration mode; specifically, when the frequency of the excitation light ω pump When the signal is close to the molecular electronic absorption center frequency ω0, the signal will be significantly enhanced; at the same time, in order to avoid excessive electronic background (such as fluorescence), ω pump Usually the non-completely resonant region (red-shift region) near ω0 is selected; in summary, the intensity of the pre-resonance stimulated Raman signal is related to the frequency detuning (ω0-ω pump ) and the electronic state line width Γ e is closely related, the best choice is usually to tune the frequency detuning to 2Γ e≤|ω0-ω pump |≤4Γ e , i.e. the electron pre-resonance range. In this example, the C≡N bond of the Rhodamine-800 sample molecule in step 1 is measured, the central wavelength of the Stokes light is fixed at 920nm, and the central wavelength of the pump light is 763nm;

[0062] Step 3, collimate the laser output by the broadband femtosecond pulse laser light source 4 in step 2 and input it into the high-resolution wavelength scanning monochromator 5, adjust the slit width and the rotation angle of the diffraction grating to output a narrow-band femtosecond pulse laser; wherein the first high-resolution wavelength scanning monochromator 501 receives the output light of the broadband femtosecond pump pulse laser light source 401, and controls its grating rotation by the electric rotating stage to realize continuous wavelength scanning; the output narrow-band pump laser is then transmitted to the time delay line 601 in the stimulated Raman signal excitation module 6; the output light of the broadband femtosecond Stokes pulse laser light source 402 is received by the second high-resolution wavelength scanning monochromator 502, and outputs a narrow-band Stokes laser, which is then transmitted to the second beam reduction system 603 in the stimulated Raman signal excitation module 6; in this example, the narrow-band laser FWHM output by the two monochromators is 0.2nm, and at the same time, the first high-resolution wavelength scanning monochromator 501 continuously scans the pump light wavelength at intervals of 0.2nm;

[0063] Step 4: The narrowband pump pulse light output by the first high-resolution wavelength scanning monochromator 501 enters the stimulated Raman signal excitation module 6, passes through the time delay line 601, the first beam reduction system 602, and the second beam splitter 608 in sequence, and is reflected by the dichroic mirror 609; at the same time, the narrowband Stokes pulse light output by the second high-resolution wavelength scanning monochromator 502 enters the stimulated Raman signal excitation module 6, passes through the second beam reduction system 603, the 1 / 4 wave plate 604, the optical modulator 605, the Glan Taylor prism 606, and the second reflector 607 in sequence, and is transmitted by the dichroic mirror 609; the two narrowband pulse laser beams combined by the dichroic mirror 609 are transmitted by the microscope objective 61 0 is used to focus on the sample placed on the sample stage 611 to stimulate stimulated Raman signals; the signal is converged by the second converging mirror 704 and filtered out by the filter 705 and the second continuous neutral density filter 706 to remove the background light and then collected by the second photodetector 707 as signal light; before the two narrow-band pulse laser beams are combined by the dichroic mirror 609, the pump light is split by the second beam splitter 608, and the reflected pump light is collected by the first photodetector 703 as reference light after passing through the first converging mirror 701 and the first continuous neutral density filter 702; during imaging, the sample is moved on the XYZ three-dimensional sample stage 611 controlled by the program to realize point scanning detection;

[0064] In the stimulated Raman scattering signal excitation module 6, the signal generator 612 outputs a modulation signal to the electro-optical modulator 605 to modulate the intensity of the Stokes light; the electric translation stage controls the movement of the first time delay line 601 to make the two light beams overlap in time dimension and finely adjust the overlap of the two light beams in space dimension at the sample 611; the sample stage 611 is moved to the focus of the objective lens 610;

[0065] Step 5: In the stimulated Raman scattering signal detection module, the voltage signal collected by the first photodetector 703 and the second photodetector 707 in step 4 is input into the phase-locked amplifier 708 for differential amplification operation and demodulation to obtain the stimulated Raman signal; the stimulated Raman signal is collected by the data acquisition card 709 controlled by Labview software, and finally the stimulated Raman spectrum measurement and imaging of the sample selected in step 1 are realized;

[0066] Before the stimulated Raman signal is measured, the Stokes light source 402 is first blocked, and the first continuous neutral density filter 702 and the second continuous neutral density filter 706 are adjusted to balance the photoelectric signals received by the first photodetector 703 and the second photodetector 707, so that the voltage signal obtained after the differential operation of the phase-locked amplifier 708 is close to 0, forming a balanced detection structure to eliminate the light path and environmental noise and improve the signal-to-noise ratio;

[0067] Step 6: tune the central wavelengths of the output lights of the broadband femtosecond pump pulse laser light source 401 and the broadband femtosecond Stokes pulse laser light source 402, and repeat steps 1 to 6 to achieve different Raman mode measurements, that is, to achieve tunable excitation stimulated Raman spectroscopy microscopy imaging.

[0068] The specific description above further illustrates the purpose, technical solutions and beneficial effects of the invention in detail. It should be understood that the above is only a specific embodiment of the present invention and is not intended to limit the scope of protection of the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A tunable stimulated Raman spectroscopy microscopy method, characterized in that: The following steps are included: Step 1: Select the sample to be tested and determine the energy level structure and Raman wave number of the Raman mode to be measured; the Raman wave number is given by the formula Calculate, where Ω represents the Raman wave number, λp represents the pump light wavelength, and λs represents the Stokes light wavelength; Step 2: The laser light emitted by the femtosecond laser (1) is split into two beams through a first beam splitter (2), one beam entering a stimulated Raman pump pulse light source (401) in a tunable broadband femtosecond pulse laser light source (4), and the other beam is reflected by a first reflector (3) and enters a stimulated Raman Stokes pulse light source (402) in the tunable broadband femtosecond pulse laser light source (4); according to the energy level structure and Raman wave number selected in step 1, the central wavelengths of the output lights of the broadband femtosecond pump pulse laser light source (401) and the broadband femtosecond Stokes pulse laser light source (402) are adjusted to achieve precise wavelength adjustment of the pump light and the Stokes light so as to match the specific Raman vibration mode of the sample; at the same time, the wavelength of the pump excitation light is tuned to a region where the absorption peak of the target sample molecule is slightly shifted toward a longer wavelength, so as to achieve an electron pre-resonance effect; this effect greatly improves the intensity of the stimulated Raman signal by enhancing the electron-vibration coupling mode of the target molecule, while reducing the interference of the background signal; Step 3, collimating the laser light outputted by the broadband femtosecond pulse laser light source (4) in step 2 and inputting it into a high-resolution wavelength scanning monochromator (5), adjusting the slit width and the rotation angle of the diffraction grating to output a narrowband femtosecond pulse laser light; The first high-resolution wavelength scanning monochromator (501) receives the output light of the broadband femtosecond pump pulse laser light source (401), and the electric rotating stage controls the rotation of its grating to achieve continuous wavelength scanning; the output narrow-band pump laser light is then transmitted to the time delay line (601) in the stimulated Raman signal excitation module (6); the output light of the broadband femtosecond Stokes pulse laser light source (402) is received by the second high-resolution wavelength scanning monochromator (502), and the narrow-band Stokes laser light is output, which is then transmitted to the second light beam reduction system (603) in the stimulated Raman signal excitation module (6); Step 4: The narrowband pump pulse light output by the first high-resolution wavelength scanning monochromator (501) enters the stimulated Raman signal excitation module (6), passes through the time delay line (601), the first beam reduction system (602), and the second beam splitter (608) in sequence, and is then reflected by the dichroic mirror (609); at the same time, the narrowband Stokes pulse light output by the second high-resolution wavelength scanning monochromator (502) enters the stimulated Raman signal excitation module (6), passes through the second beam reduction system (603), the 1 / 4 wave plate (604), the optical modulator (605), the Glan Taylor prism (606), and the second reflector (607) in sequence, and is then transmitted by the dichroic mirror (609); the two narrowband pulse laser beams combined by the dichroic mirror (609) are transmitted by the microscope. The objective lens (610) is used to focus on the sample placed on the sample stage (611) to stimulate the stimulated Raman signal; the signal is converged by the second converging mirror (704) and filtered by the filter (705) and the second continuous neutral density filter (706) to remove the background light, and then collected by the second photodetector (707) as signal light; before the two narrow-band pulse laser beams are combined by the dichroic mirror (609), the pump light is split by the second beam splitter (608), and the reflected pump light passes through the first converging mirror (701) and the first continuous neutral density filter (702) and is collected by the first photodetector (703) as reference light; during imaging, the sample is moved on the XYZ three-dimensional sample stage (611) controlled by the program to achieve point scanning; Step 5: input the voltage signals collected by the first photodetector (703) and the second photodetector (707) in step 4 into a phase-locked amplifier (708) for differential amplification and demodulation to obtain the stimulated Raman signal; the stimulated Raman signal is collected by a data acquisition card (709) controlled by Labview software to achieve stimulated Raman spectrum measurement and imaging of the sample selected in step 1; Step 6: Tuning the central wavelengths of the output lights of the broadband femtosecond pump pulse laser light source (401) and the broadband femtosecond Stokes pulse laser light source (402), repeating steps 1 to 6 to achieve different Raman mode measurements, that is, achieving tunable excitation stimulated Raman spectroscopy microscopy imaging.

2. The method for tunable stimulated Raman spectroscopy microscopy according to claim 1, characterized in that: The time delay line (601) is arranged between the first high-resolution wavelength scanning monochromator (501) and the first beam reduction system (602) in the pump pulse optical path, and is used to change the optical path difference between the pump light and the Stokes light, so that the pump pulse and the Stokes pulse are aligned in the time dimension; The first beam reduction system (602) and the second beam reduction system (603) are used to spatially reduce the two pulsed light beams to couple the light modulator (605) with the aperture of the microscope objective lens (610); The quarter wave plate (604) is used to convert the Stokes light from linear polarization to elliptical polarization; The signal generator (612) outputs a modulation signal, which is amplified by a voltage amplifier (613) and then sent to an optical modulator (605), where the intensity of the pulsed light is modulated by the optical modulator (605); The Glan Taylor prism (606) is used to change the modulated light from elliptical polarization to linear polarization; The light intensity modulation system composed of a quarter wave plate (604), a light modulator (605) and a Glan-Taylor prism (606) is placed between the beam reduction system of the pump light path or the Stokes light path and the dichroic mirror (609); The dichroic mirror (609) is used to spatially combine the Stokes light and the narrow-band pump light so that the pump pulse and the Stokes pulse are aligned in the spatial dimension; The signal generator (612) and the data acquisition card (709) are synchronously triggered by a laser; the signal generator (612) inputs a signal with the same frequency as the modulation signal into the phase-locked amplifier (708) and is synchronously triggered.

3. The method for tunable stimulated Raman spectroscopy microscopy according to claim 1, characterized in that: Before measuring the stimulated Raman signal, the Stokes light source (402) is first blocked, and the first continuous neutral density filter (702) and the second continuous neutral density filter (706) are adjusted to balance the photoelectric signals received by the first photodetector (703) and the second photodetector (707), so that the voltage signal obtained after the differential operation of the phase-locked amplifier (708) is close to 0, forming a balanced detection structure to eliminate optical path noise and improve the signal-to-noise ratio.

4. A tunable excitation stimulated Raman spectroscopy microscopy imaging device, used to implement the tunable excitation stimulated Raman spectroscopy microscopy imaging method as claimed in claims 1, 2 and 3, characterized in that: It includes a wide-band adjustable wavelength laser light source module, a high-resolution wavelength scanning module, a stimulated Raman signal excitation module and a stimulated Raman signal detection module; A wide-band tunable laser light source module based on an optical parametric amplifier, which is used to output two broadband femtosecond pulse lasers with different wavelengths and continuously adjustable wavelengths; The wide-band tunable laser light source module is composed of a femtosecond laser (1), a first beam splitter (2), a first reflector (3) and a tunable broadband femtosecond pulse laser light source (4); A high-resolution wavelength scanning module is used to achieve spectral dispersion of laser outputs of a broadband femtosecond pump pulse laser light source (401) and a broadband femtosecond Stokes pulse laser light source (402), and output two narrow-band femtosecond pulse lights; it is composed of a first high-resolution wavelength scanning monochromator (501) and a second high-resolution wavelength scanning monochromator (502); A stimulated Raman scattering signal excitation module is used to focus two beams of narrow-band femtosecond pulse light with temporal and spatial overlap on a sample and excite a stimulated Raman signal; it is composed of a time delay line (601), a first beam reduction system (602), a second beam reduction system (603), a quarter wave plate (604), an optical modulator (605), a Glan Taylor prism (606), a second reflector (607), a second beam splitter (608), a dichroic mirror (609), a microscope objective (610), a sample stage (611), a signal generator (612), and a voltage amplifier (613); The stimulated Raman signal detection module is used to collect the stimulated Raman scattering signal and convert it into an electrical signal, and then perform data acquisition after demodulating the signal using a phase-locked amplifier; the module is composed of a second beam splitter (608), a first converging mirror (701), a first continuous neutral density filter (702), a first photodetector (703), a second converging mirror (704), a short-wave pass filter (705), a second continuous neutral density filter (706), a second photodetector (707), a signal generator (612), a phase-locked amplifier (708) and a data acquisition card (709).

5. The tunable stimulated Raman spectroscopy microscopy imaging device according to claim 4, characterized in that: The first high-resolution wavelength scanning monochromator (501) and the second high-resolution wavelength scanning monochromator (502) have the same structure, and are composed of a third reflector (8), a plane diffraction blazed grating (9), a cylindrical mirror (10), an adjustable slit (11) and a roof mirror (12) to form a 4f system; after the broadband pulse lasers output by the stimulated Raman pump pulse light source (401) and the stimulated Raman Stokes pulse light source (402) enter the first high-resolution wavelength scanning monochromator (501) and the second high-resolution wavelength scanning monochromator (502) respectively, they are reflected by the third reflector (8) to the plane diffraction blazed grating (9) for dispersion and splitting, wherein the first-order diffraction light is converged by the cylindrical mirror (10) and output as narrow-band femtosecond pulse light through the adjustable slit (11), and the pulse light is reflected back by the roof mirror (12) and returns along the original optical path, thereby ensuring that the optical path structure is not changed during the wavelength scanning.

6. The tunable stimulated Raman spectroscopy microscopy imaging device according to claim 4, characterized in that: The optical modulator (605) is an electro-optic modulator or an acousto-optic modulator, and is used to modulate the intensity of the laser. If the light intensity modulation system composed of a quarter wave plate (604), an optical modulator (605) and a Glan-Taylor prism (606) is placed between the first beam reduction system (602) and the dichroic mirror (609) in the pump light path, the stimulated Raman gain signal is detected, and at this time, the filter (705) is a long-wave pass filter used to filter out excess pump light noise; If the light intensity modulation system composed of a 1 / 4 wave plate (604), an optical modulator (605) and a Glan-Taylor prism (606) is placed between the second light beam reduction system (603) and the dichroic mirror (609) in the Stokes light path, the stimulated Raman loss signal is detected. At this time, the filter (705) is a short-wave pass filter used to filter out excess Stokes light noise.

Images