Tunable Excitation Stimulated Raman Spectroscopy Microscopic Imaging Method and Apparatus

Abstract

A tunable stimulated Raman spectroscopy microscopy imaging method and apparatus belongs to the field of spectral detection and optical imaging. The apparatus of this invention includes a wide-band tunable 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 focuses two temporally and spatially overlapping narrowband femtosecond pulses onto the sample to excite a stimulated Raman signal, while simultaneously moving the sample to achieve point scanning microscopy imaging. The stimulated Raman signal detection module converts the stimulated Raman scattering signal into an electrical signal, demodulates it using a lock-in amplifier, and then records the spectral information using a data acquisition card. This invention utilizes the wide-band tunable characteristics of an optical parametric amplifier to tune the pump light to the long-wavelength region of the sample's absorption peak, achieving electron pre-resonance stimulated Raman; and combines balanced detection technology at the detection end to achieve high sensitivity, high resolution, and high signal-to-noise ratio detection of the stimulated Raman signal.

Description

Technical Field

[0001] This invention relates to a tunable excitation stimulated Raman spectroscopy microscopic imaging method and apparatus, belonging to the field of spectral detection and optical imaging technology. Background Technology

[0002] Stimulated Raman spectroscopy (SRS) is a third-order nonlinear optical process used to study the vibrational properties of molecules. SRS involves the interaction of two photons and a molecule. When a pump photon and a Stokes photon simultaneously irradiate a molecule, if the energy difference between the two photons equals the vibrational energy level difference of the molecule, the molecule is excited to a dummy energy level and then rapidly descends to its vibrational level. This results in an enhancement of the Stokes photon and a decay of the pump photon. Therefore, the SRS signal can be obtained by detecting changes in the intensity of either the pump photon or the Stokes photon.

[0003] Although stimulated Raman spectroscopy has become increasingly sophisticated, the stimulated Raman scattering process is often exceptionally weak, especially in low-concentration samples. Obtaining a sufficiently strong signal may require increasing laser power or performing multiple scans, further increasing experimental time and complexity. Furthermore, Raman scattering has low detection sensitivity and is easily affected by fluorescence background. On the other hand, most existing techniques use ultrafast lasers based on optical parametric oscillators (OPOs), while Stokes light is the direct output of the laser, which significantly limits the practical application of stimulated Raman scattering. Further research is needed to address the challenges of narrow excitation wavelength ranges, limited sample types that can be measured, and low signal-to-noise ratios in existing methods, and to achieve efficient excitation of Raman vibrational states, thereby expanding the application of stimulated Raman spectroscopy to a wider range of fields. Summary of the Invention

[0004] To address the problems of low signal-to-noise ratio, limited wavelength adjustment range, and insufficient detection sensitivity in existing stimulated Raman spectroscopy detection systems, the present invention aims to provide a tunable stimulated Raman spectroscopy microscopic imaging method and apparatus. By utilizing the wide-band tunable characteristics of an optical parametric amplifier, the excitation wavelength is tuned to the electronic pre-resonance range. Combined with balanced detection technology at the detection end, it is possible to achieve high sensitivity, high resolution, and high signal-to-noise ratio detection of stimulated Raman scattering signals.

[0005] The objective of this invention is achieved through the following technical solution:

[0006] This invention discloses a tunable excitation stimulated Raman spectroscopy microscopy imaging method, comprising the following steps:

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

[0008] Step 2: The laser emitted by the femtosecond laser is split into two beams by the first beam splitter. One beam enters the stimulated Raman pump pulse source in the tunable broadband femtosecond pulse laser source, and the other beam is reflected by the first mirror and enters the stimulated Raman Stokes pulse source in the tunable broadband femtosecond pulse laser source. Based on the energy level structure and Raman wavenumber selected in Step 1, the center wavelengths of the output light from the broadband femtosecond pump pulse laser source and the broadband femtosecond Stokes pulse laser source are adjusted to achieve precise wavelength tuning of the pump light and Stokes light, matching them to the 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 sample molecule is slightly shifted to a longer wavelength to achieve the electronic pre-resonance effect. This effect significantly increases the intensity of the stimulated Raman signal by enhancing the electronic-vibrational coupling mode of the target molecule, while reducing interference from background signals such as fluorescence.

[0009] Step 3: After collimating the laser output from the broadband femtosecond pulsed laser source in Step 2, input it into a high-resolution wavelength scanning monochromator. Adjust the slit width and the rotation angle of the diffraction grating to output a narrowband femtosecond pulsed laser. The first high-resolution wavelength scanning monochromator receives the output light from the broadband femtosecond pump pulsed laser source, and its grating is rotated by an electric rotary stage to achieve continuous wavelength scanning. The output narrowband pump laser is then transmitted to the time delay line in the stimulated Raman signal excitation module. The output light from the broadband femtosecond Stokes pulsed laser source is received by the second high-resolution wavelength scanning monochromator, which outputs a narrowband Stokes laser, which is then transmitted to the second beam constriction system in the stimulated Raman signal excitation module.

[0010] Step 4: The narrowband pump pulse light output from the first high-resolution wavelength scanning monochromator enters the stimulated Raman signal excitation module, and after passing through the time delay line, the first beam shrinking system, and the second beam splitter, it is reflected by the dichroic mirror. Simultaneously, the narrowband Stokes pulse light output from the second high-resolution wavelength scanning monochromator enters the stimulated Raman signal excitation module, and after passing through the second beam shrinking system, the quarter-wave plate, the optical modulator, the Glan-Taylor prism, and the second reflecting mirror, it is transmitted through the dichroic mirror. The narrowband pulsed laser beams combined by the dichroic mirror are then focused by the microscope objective. Stimulated Raman signals are excited on the sample placed on the sample stage. The signal is converged by a second converging mirror and filtered by a filter and a second continuous neutral density filter to remove background light before being collected by a second photodetector as signal light. Before the two narrowband pulsed laser beams are combined by a dichroic mirror, the pump light is split by a second beam splitter. One of the reflected pump lights passes through a first converging mirror and a first continuous neutral density filter before being collected by a first photodetector as reference light. During imaging, the sample is moved by a program-controlled XYZ three-dimensional sample stage to achieve point scanning.

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

[0012] Step 6: Tune the center wavelength of the output light from the broadband femtosecond pumped pulsed laser source (401) and the broadband femtosecond Stokes pulsed laser 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.

[0013] Furthermore, the time delay line is disposed between the first high-resolution wavelength scanning monochromator and the first beam-shrinking 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 and second beam-shrinking systems are used to spatially shrink the two pulse beams to couple the light modulator to the microscope objective aperture.

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

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

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

[0018] Furthermore, the light intensity modulation system, consisting of a quarter-wave plate, a light modulator, and a Glan Taylor prism, is placed between the beam-shrinking 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 narrowband pump light, aligning the pump pulse and the Stokes pulse in spatial dimensions.

[0020] Furthermore, the signal generator and the data acquisition card are synchronously triggered by the laser; the signal generator inputs a signal with the same frequency as the modulation signal to the lock-in amplifier and triggers it synchronously.

[0021] Furthermore, before the stimulated Raman signal measurement, the Stokes light source is blocked, and the first continuous neutral density filter and the second continuous neutral density filter are adjusted to balance the photoelectric signals received by the first photodetector and the second photodetector. This makes the voltage signal obtained after differential operation by the lock-in amplifier close to 0, thus forming a balanced detection structure to eliminate optical path noise and improve the signal-to-noise ratio.

[0022] This invention discloses a tunable excitation stimulated Raman spectroscopy microscopy imaging device for realizing a tunable excitation stimulated Raman spectroscopy microscopy imaging method. The tunable excitation stimulated Raman spectroscopy microscopy imaging device includes a broadband tunable wavelength laser source module, a high-resolution wavelength scanning module, a stimulated Raman signal excitation module, and a stimulated Raman signal detection module.

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

[0024] The high-resolution wavelength scanning module is used to perform spectral dispersion on the output lasers of the broadband femtosecond pump pulse laser source and the broadband femtosecond Stokes pulse laser source, and output two narrowband femtosecond pulse beams; it consists 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 time- and spatially overlapping narrowband femtosecond pulses onto the sample and excite stimulated Raman signals. It consists of a time delay line, a first beam shrinking system, a second beam shrinking system, a quarter-wave plate, an optical modulator, a Glan Taylor prism, a second reflecting mirror, 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 then perform data acquisition after demodulating the signal using a lock-in amplifier; it consists of a second beam splitter, a first converging mirror, a first continuous neutral density filter, a first photodetector, a second converging mirror, a short-pass filter, a second continuous neutral density filter, a second photodetector, a signal generator, a lock-in amplifier, and a data acquisition card.

[0027] Furthermore, the first and second high-resolution wavelength scanning monochromators have the same structure, consisting of a third mirror, a plane diffraction blazed grating, a cylindrical mirror, an adjustable slit, and a roof mirror forming a 4f system. The broadband pulsed lasers output from the stimulated Raman pumped pulse source and the stimulated Raman Stokes pulse source enter the first and second high-resolution wavelength scanning monochromators, respectively, and are reflected by the third mirror onto the plane diffraction blazed grating for dispersion. The first-order diffracted light is converged by the cylindrical mirror and outputs a narrowband femtosecond pulse light through the adjustable slit. This pulse light is reflected back by the roof mirror and returns along the original optical path, ensuring that the optical path structure is not changed during wavelength scanning.

[0028] Furthermore, an electro-optic modulator or an acousto-optic modulator is selected as the optical modulator to modulate the intensity of the laser.

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

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

[0031] Beneficial effects:

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

[0033] 2. The tunable excitation stimulated Raman spectroscopy microscopy imaging method and apparatus disclosed in this 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 during the detection process, and enhance the weak signal detection capability of the system.

[0034] 3. The tunable excitation stimulated Raman spectroscopy microscopy imaging method and apparatus disclosed in this invention tunes the pump excitation wavelength to a region where the absorption peak of the sample molecule is slightly shifted to a longer wavelength to achieve an electronic pre-resonance effect. This effect can significantly improve the intensity of the stimulated Raman signal by enhancing the electronic-vibrational coupling mode of the target molecule, while reducing the interference of the background signal.

[0035] 4. The tunable excitation stimulated Raman spectroscopy microscopic imaging method and apparatus disclosed in this invention, wherein the high-resolution wavelength scanning monochromator consists of a third reflecting mirror, a plane diffraction blazed grating, a cylindrical mirror, an adjustable slit, and a roof mirror forming a 4f system; the first-order diffracted light after dispersion on the plane diffraction grating is converged by the cylindrical mirror and output as a narrowband femtosecond pulse light through the adjustable slit. The pulse light returns along the original optical path after being reflected back by the roof mirror, ensuring that the optical path structure is not changed during wavelength scanning. Attached Figure Description

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

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

[0038] Wherein: 1-Femtosecond laser, 2-First beam splitter, 3-First reflector, 4-Wideband tunable laser source, 401-Wideband tunable wavelength pump source, 402-Wideband tunable wavelength Stokes 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 constriction system, 603-Second beam constriction system, 604-Quarter-wave plate, 605 - Electro-optic modulator, 606-Glan Taylor prism, 607-Second reflecting mirror, 608-Second beam splitter, 609-Dichroic mirror, 610-Microscopic objective lens, 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-Lock-in amplifier, 709-Data acquisition card.

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

[0040] Among them: 8-Third reflecting mirror, 9-Planar diffraction blazed grating, 10-Cylindrical mirror, 11-Adjustable slit, 12-Roof mirror. Detailed Implementation

[0041] To better illustrate the purpose and advantages of the present invention, the invention will be further described below in conjunction with the accompanying drawings and examples.

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

[0043] Example 1:

[0044] like Figure 1 As shown in the schematic diagram of the logical structure of the tunable stimulated Raman spectroscopy microscopy imaging method provided in this embodiment of the invention, a broadband laser is generated by a broadband tunable femtosecond pump source and a broadband tunable femtosecond Stokes source, and then a narrowband laser is generated by dispersion by a high-resolution wavelength scanning monochromator; the two temporally and spatially overlapping narrowband lasers are focused at the sample to excite the electron pre-resonance stimulated Raman signal, and the sample is moved to achieve point scanning tunable stimulated Raman spectroscopy microscopy imaging.

[0045] like Figure 2 The diagram shows an embodiment of the tunable stimulated Raman spectroscopy microscopic imaging device provided in this invention. The device described in this example measures stimulated Raman loss signals and includes a femtosecond laser 1, a first beam splitter 2, a first mirror 3, a wideband tunable laser source 4, a wideband tunable wavelength pump source 401, a wideband tunable wavelength Stokes 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 shortening system 602, a second beam shortening system 603, and a quadrupole... The system includes: a waveplate 604, an electro-optic modulator 605, a Glan Taylor prism 606, a second reflecting mirror 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 short-pass filter 705, a second continuous neutral density filter 706, a second photodetector 707, a lock-in 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 this embodiment of the invention is shown. The two high-resolution wavelength scanning monochromators have the same structure and are composed of a third reflecting mirror 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.

[0047] Broadband pulsed lasers output from stimulated Raman pumped pulsed light source 401 and stimulated Raman Stokes pulsed light source 402 enter the first high-resolution wavelength scanning monochromator 501 and the second high-resolution wavelength scanning monochromator 502, respectively. They are then reflected by the third mirror 8 onto the plane diffraction blazed grating 9 controlled by the electric rotary stage for dispersion. The first-order diffracted light is converged by the cylindrical mirror 10 and output as a narrowband femtosecond pulsed laser through the adjustable slit 11. The narrowband femtosecond pulsed light is reflected back by the roof mirror 12 and returns along the original optical path, ensuring that the optical path structure is not changed during wavelength scanning.

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

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

[0050] The wideband tunable laser source 4 generates a wideband tunable wavelength laser source that can cover a wide wavelength range (600nm-950nm) from visible to near infrared, with a half width at half maximum (WHM) of 8nm and a spot diameter of 4mm, enabling the system to detect the Raman vibration modes of a variety of molecules.

[0051] The grating 9 is a planar diffraction blazed grating with 1200 lines / mm and a center wavelength of 750nm; the cylindrical mirror 10 has a focal length of 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-shrinking system 602 and the second beam-shrinking system 603 have a beam-shrinking ratio of 2:1, which is used to shrink the Stokes spot size to 2 mm to couple the light modulator 605 to the microscope objective 610.

[0053] The optical modulator 605 is an electro-optic modulator with an inlet diameter of 2mm. In this example, it is placed between the second beam-shrinking 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-optic modulator 605 to modulate the Stokes light intensity, with a modulation efficiency of >95%.

[0055] The filter 705 is a short-pass filter with a cutoff wavelength of 850nm;

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

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

[0058] Step 2: The laser emitted by the femtosecond laser 1 is split into two beams by the first beam splitter 2. One beam enters the stimulated Raman pump pulse source 401 in the tunable broadband femtosecond pulse laser source 4, and the other beam is reflected by the first mirror 3 and enters the stimulated Raman Stokes pulse source 402 in the tunable broadband femtosecond pulse laser source 4. According to the energy level structure and Raman wavenumber selected in Step 1, the center wavelengths of the output light from the broadband femtosecond pump pulse laser source 401 and the broadband femtosecond Stokes pulse laser source 402 are adjusted to achieve precise wavelength tuning of the pump light and Stokes light, matching them to 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 to a longer wavelength to achieve an electronic pre-resonance effect. This effect significantly increases the intensity of the stimulated Raman signal by enhancing the electronic-vibrational coupling mode of the target molecule, while reducing the interference of the background signal.

[0059] The electron pre-resonance effect is achieved according to the following equation:

[0060]

[0061] In the formula, σ is the electronic pre-resonance Raman cross section; ω0 is the electron absorption center frequency of the molecule, i.e., the resonance frequency; ω pump The frequency of the excitation light (pump light); Γ e ω is the linewidth of the molecular electronic states (the width of the absorption band); K is a constant related to molecular properties; vib It is the frequency of the vibration mode; specifically, when the frequency ω of the excitation light... pump The signal is significantly enhanced when the frequency approaches the molecular electron absorption center frequency ω0; at the same time, to avoid excessive electronic background (such as fluorescence), ω pump Typically, the incomplete resonance region (redshift region) near ω0 is chosen; in summary, the pre-resonance stimulated Raman signal intensity is related to the frequency detuning (ω0-ω). pump ) and electronic state linewidth Γ e Closely related, the best choice is usually to tune the frequency detuning to 2Γ. e≤|ω0-ω pump |≤4Γ e This refers to the electronic pre-resonance range. In this example, to measure the C≡N bonds in the Rhodamine-800 sample molecule in step one, the Stokes light center wavelength is fixed at 920 nm, and the pump light center wavelength is 763 nm.

[0062] Step 3: After collimating the laser output from the broadband femtosecond pulsed laser source 4 in Step 2, 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 narrowband femtosecond pulsed laser. The first high-resolution wavelength scanning monochromator 501 receives the output light from the broadband femtosecond pump pulsed laser source 401 and controls its grating rotation by an electric rotary stage to achieve continuous wavelength scanning. The output narrowband pump laser is then transmitted to the time delay line 601 in the stimulated Raman signal excitation module 6. The output light from the broadband femtosecond Stokes pulsed laser source 402 is received by the second high-resolution wavelength scanning monochromator 502, which outputs a narrowband Stokes laser, which is then transmitted to the second beam shrinking system 603 in the stimulated Raman signal excitation module 6. In this example, the narrowband laser FWHM output by the two monochromators is 0.2nm, while the first high-resolution wavelength scanning monochromator 501 continuously scans the pump light wavelength at 0.2nm intervals.

[0063] Step 4: The narrowband pump pulse light output from the first high-resolution wavelength scanning monochromator 501 enters the stimulated Raman signal excitation module 6, and passes sequentially through the time delay line 601, the first beam shrinking system 602, and the second beam splitter 608 before being reflected by the dichroic mirror 609. Simultaneously, the narrowband Stokes pulse light output from the second high-resolution wavelength scanning monochromator 502 enters the stimulated Raman signal excitation module 6, and passes sequentially through the second beam shrinking system 603, the quarter-wave plate 604, the optical modulator 605, the Glan-Taylor prism 606, and the second reflecting mirror 607 before being transmitted through the dichroic mirror 609. The narrowband pulsed laser beam resulting from the combination of the two beams through the dichroic mirror 609 is then transmitted through the microscope objective 61. The 0 is used to focus on the sample placed on the sample stage 611 to excite a stimulated Raman signal; this 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 background light before being collected by the second photodetector 707 as the signal light; before the two narrowband pulsed 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 after passing through the first converging mirror 701 and the first continuous neutral density filter 702 as the reference light; during imaging, the sample is moved on the XYZ three-dimensional sample stage 611 under program control to achieve point scanning detection;

[0064] In the stimulated Raman scattering signal excitation module 6, the signal generator 612 outputs a modulation signal to the electro-optic modulator 605 to modulate the Stokes light intensity; the first time delay line 601 is moved by the electric displacement stage to make the time dimension of the two beams overlap and finely adjust the spatial dimension overlap of the two beams at the sample 611; the sample stage 611 is moved to the focal point of the objective lens 610.

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

[0066] Before the stimulated Raman signal measurement, the Stokes light source 402 is 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 differential operation of the lock-in amplifier 708 is close to 0, thus forming a balanced detection structure to eliminate optical path and environmental noise and improve the signal-to-noise ratio.

[0067] Step 6: Tune the center wavelength of the output light from the broadband femtosecond pumped pulsed laser source 401 and the broadband femtosecond Stokes pulsed laser 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 above detailed description further illustrates the purpose, technical solution, and beneficial effects of the invention. It should be understood that the above description 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 within the scope of protection of the present invention.

Claims

1. A tunable excitation stimulated Raman spectroscopy microscopic imaging method, characterized in that: Includes the following steps, Step 1: Select the sample to be tested and determine the energy level structure and Raman wavenumber of the Raman mode to be measured; the Raman wavenumber is given by the formula... The calculation is performed, where Ω represents the Raman wavenumber, λp represents the pump light wavelength, and λs represents the Stokes light wavelength. Step 2: The laser emitted by the femtosecond laser (1) is split into two beams by the first beam splitter (2). One beam enters the broadband femtosecond pump pulse laser source (401) in the tunable broadband femtosecond pulse laser source (4), and the other beam is reflected by the first mirror (3) into the broadband femtosecond Stokes pulse laser source (402) in the tunable broadband femtosecond pulse laser source (4). According to the energy level structure and Raman wavenumber selected in Step 1, the center wavelength of the output light of the broadband femtosecond pump pulse laser source (401) and the broadband femtosecond Stokes pulse laser source (402) is adjusted to achieve precise wavelength adjustment of the pump light and 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 the region where the absorption peak of the target sample molecule is slightly shifted to a longer wavelength to achieve the electronic pre-resonance effect. This effect enhances the electronic-vibrational coupling mode of the target molecule, greatly increases the intensity of the stimulated Raman signal, and reduces the interference of the background signal. Step 3: After collimating the laser output from the broadband femtosecond pulse laser source (4) in Step 2, input it into the high-resolution wavelength scanning monochromator (5), and adjust the slit width and the rotation angle of the diffraction grating to output a narrowband femtosecond pulse laser; wherein the first high-resolution wavelength scanning monochromator (501) receives the output light from the broadband femtosecond pump pulse laser source (401), and its grating is rotated by an electric rotary stage to achieve continuous wavelength scanning; the output narrowband pump laser is then transmitted to the time delay line (601) in the stimulated Raman signal excitation module (6); the output light from the broadband femtosecond Stokes pulse laser source (402) is received by the second high-resolution wavelength scanning monochromator (502), and a narrowband Stokes laser is output, which is then transmitted to the second beam contraction system (603) in the stimulated Raman signal excitation module (6); Step 4: The narrowband pump pulse light output from 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 shrinking system (602), and the second beam splitter (608) in sequence, and is reflected by the dichroic mirror (609); simultaneously, the narrowband Stokes pulse light output from the second high-resolution wavelength scanning monochromator (502) enters the stimulated Raman signal excitation module (6), passes through the second beam shrinking system (603), the quarter-wave plate (604), the optical modulator (605), the Glan Taylor prism (606), and the second reflecting mirror (607) in sequence, and is transmitted by the dichroic mirror (609); the narrowband pulse laser beam after being combined by the dichroic mirror (609) is then transmitted through the display. The micro-objective (610) is used to focus on the sample placed on the sample stage (611) to excite 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 is collected by the second photodetector (707) as the signal light; before the two narrowband pulsed laser beams are combined by the dichroic mirror (609), the pump light is split by the second beam splitter (608), and one of the reflected pump lights is collected by the first photodetector (703) as the reference light after passing through the first converging mirror (701) and the first continuous neutral density filter (702); during imaging, the sample is moved by 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 the lock-in amplifier (708) for differential amplification and demodulation to obtain the stimulated Raman signal; the stimulated Raman signal is acquired by the data acquisition card (709) controlled by LabVIEW software to realize the stimulated Raman spectroscopy measurement and imaging of the sample selected in Step 1; Step 6: Tune the center wavelength of the output light from the broadband femtosecond pumped pulsed laser source (401) and the broadband femtosecond Stokes pulsed laser 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.

2. The tunable excitation stimulated Raman spectroscopy microscopy imaging method as described in claim 1, characterized in that: The time delay line (601) is disposed between the first high-resolution wavelength scanning monochromator (501) and the first beam shrinking 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 shrinking system (602) and the second beam shrinking system (603) are used to spatially shrink two pulse beams to couple the light modulator (605) to the aperture of the microscope objective (610); The quarter-wave plate (604) is used to convert Stokes light from linear polarization to elliptic polarization; The signal generator (612) outputs a modulation signal, which is amplified by the voltage amplifier (613) and sent to the optical modulator (605), which modulates the intensity of the pulsed light. The Glan Taylor prism (606) is used to change the modulated light from elliptic polarization to linear polarization; A light intensity modulation system consisting of a quarter wave plate (604), an optical modulator (605) and a Glan Taylor prism (606) is placed between the beam-shrinking system of the pump optical path or the Stokes optical path and the dichroic mirror (609). The dichroic mirror (609) is used to spatially combine the Stokes light and the narrowband pump light, so that the pump pulse and the Stokes pulse are aligned in spatial dimension. The signal generator (612) and the data acquisition card (709) are synchronously triggered by the laser; the signal generator (612) inputs a signal with the same frequency as the modulation signal to the lock-in amplifier (708) and triggers it synchronously.

3. The tunable excitation stimulated Raman spectroscopy microscopy imaging method as described in claim 1, characterized in that: Before the stimulated Raman signal measurement, the broadband femtosecond Stokes pulse laser source (402) is blocked. 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 differential operation of the lock-in amplifier (708) is close to 0, thus 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 realize the tunable excitation stimulated Raman spectroscopy microscopy imaging method as described in claim 1, 2 or 3, characterized in that: It includes a wide-band tunable wavelength laser source module, a high-resolution wavelength scanning module, a stimulated Raman signal excitation module, and a stimulated Raman signal detection module; A wideband tunable laser source module based on an optical parametric amplifier is used to output two broadband femtosecond pulse lasers with different wavelengths and continuously adjustable wavelengths. The wideband tunable laser source module consists of a femtosecond laser (1), a first beam splitter (2), a first reflector (3), and a tunable broadband femtosecond pulse laser source (4). The high-resolution wavelength scanning module is used to perform spectral dispersion on the output lasers of the broadband femtosecond pumped pulse laser source (401) and the broadband femtosecond Stokes pulse laser source (402) to output two narrowband femtosecond pulses; it consists of a first high-resolution wavelength scanning monochromator (501) and a second high-resolution wavelength scanning monochromator (502). The stimulated Raman scattering signal excitation module is used to focus two time- and spatially overlapping narrowband femtosecond pulses onto the sample and excite stimulated Raman signals; it consists of a time delay line (601), a first beam shrinking system (602), a second beam shrinking system (603), a quarter wave plate (604), an optical modulator (605), a Glan Taylor prism (606), a second mirror (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 lock-in amplifier; it consists 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-pass filter (705), a second continuous neutral density filter (706), a second photodetector (707), a signal generator (612), a lock-in amplifier (708), and a data acquisition card (709).

5. The tunable excitation stimulated Raman spectroscopy microscopy imaging device as described in 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. They are composed of a third mirror (8), a plane diffraction blazed grating (9), a cylindrical mirror (10), an adjustable slit (11), and a roof mirror (12) forming a 4f system. The broadband pulsed lasers output from the broadband femtosecond pumped pulsed laser source (401) and the broadband femtosecond Stokes pulsed laser source (402) enter the first high-resolution wavelength scanning monochromator (501) and the second high-resolution wavelength scanning monochromator (502) respectively. They are then reflected by the third mirror (8) onto the plane diffraction blazed grating (9) for dispersion. The first-order diffracted light is converged by the cylindrical mirror (10) and outputs a narrowband femtosecond pulsed light through the adjustable slit (11). The pulsed light is reflected back by the roof mirror (12) and returns along the original optical path, ensuring that the optical path structure is not changed during wavelength scanning.

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

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