Coherent anti-Stokes Raman scattering spectroscopy measurement and microscopic imaging apparatus and method

Abstract

The present invention belongs to the technical field of Raman scattering microscopy imaging, and particularly relates to a coherent anti-Stokes Raman scattering spectroscopy measurement and microscopy imaging device and method, which includes a femtosecond pulse laser. The laser pulses generated by the femtosecond pulse laser are sequentially irradiated on a sample to be measured placed on a two-dimensional precision translation stage after passing through a first mirror, a first prism pair, the edge of a blade, a second prism pair, a second mirror, a resonant galvanometer scanner and an objective lens, and scattered signals are formed. The blade is used to generate a steep edge of the laser spectrum by means of occlusion. The scattered signals pass through the transmission part of the sample to be measured and then pass through a condenser lens and an ultra-steep short-pass filter, and then are split into two paths by a notch filter. One path enters a first spectrometer through a lens for coherent anti-Stokes Raman scattering spectroscopy measurement; the other path enters a lock-in amplifier after passing through a photomultiplier tube for coherent anti-Stokes Raman scattering microscopy imaging. The overall structure of the device of the present invention is simple and can achieve rapid imaging.

Description

Technical Field

[0001] The present invention belongs to the technical field of Raman scattering microscopy, and particularly relates to a coherent anti-Stokes Raman scattering spectrum measurement and microscopy device and method. Background Art

[0002] Compared with ordinary multi-beam coherent anti-Stokes Raman scattering devices and single-beam coherent anti-Stokes Raman scattering devices implemented based on a pulse shaper, the single-beam coherent anti-Stokes Raman scattering device implemented based on a notch filter has the advantages of simple and compact structure; wherein, for the single-beam coherent anti-Stokes Raman scattering device implemented based on a notch filter, it generates a notch feature on the laser spectrum and a similar feature on the coherent anti-Stokes Raman scattering spectrum; wherein, due to the small size and simplicity of the notch filter, it is easy to be installed on a galvanometer scanner to perform high-frequency modulation on the laser; secondly, since the position of the resonance signal is only related to the notch frequency position, the non-resonance signal is used as a local oscillator by using the lock-in amplification method, and the non-resonance signal is removed while the weak resonance signal is amplified, and thus it can be used for all coherent anti-Stokes Raman scattering schemes implemented based on fiber components, and it can be easily extended to realize low-frequency CARS spectra and microscopes to be applicable to the study of low-frequency vibration modes of large biomolecules.

[0003] However, in the single-beam coherent anti-Stokes Raman scattering device implemented based on a notch filter, due to the limited modulation speed of the galvanometer scanner used in the device, the single-pixel imaging speed is generally limited to the millisecond level; currently, in order to detect the coherent anti-Stokes Raman scattering signal in the low-frequency band, it is usually necessary to use a combination of an ultra-steep long-pass filter and an ultra-steep short-pass filter to achieve, and the introduction of the ultra-steep long-pass filter further increases the system cost and at the same time increases the complexity of the system structure. Summary of the Invention

[0004] Aiming at the technical problems existing in the prior art, the present invention provides a coherent anti-Stokes Raman scattering spectrum measurement and microscopy device and method to solve the technical problems that in the single-beam coherent anti-Stokes Raman scattering device implemented based on a notch filter, the system cost is further increased and the complexity of the system structure is increased due to the introduction of the ultra-steep long-pass filter.

[0005] To achieve the above object, the technical solution adopted by the present invention is as follows:

[0006] The present invention provides a coherent anti-Stokes Raman scattering spectroscopy measurement and microscopic imaging device, including a femtosecond pulsed laser. The laser pulses generated by the femtosecond pulsed laser are sequentially irradiated on a sample to be measured placed on a two-dimensional precision translation stage after passing through a first mirror, a first prism pair, the edge of a blade, a second prism pair, a second mirror, a resonant galvanometer scanner, and an objective lens, and scattering signals are formed. Among them, the blade is used to generate a steep edge of the laser spectrum by means of occlusion.

[0007] The scattering signals pass through the transmitted part of the sample to be measured and then pass through a condenser lens and an ultrasharp short-pass filter, and then are split into two paths by a notch filter. One path enters a first spectrometer through a lens for coherent anti-Stokes Raman scattering spectroscopy measurement; the other path enters a lock-in amplifier after passing through a photomultiplier tube for coherent anti-Stokes Raman scattering microscopic imaging.

[0008] Further, the thickness of the blade is 0.2 - 0.6 mm, and the length of the blade is 10 - 50 mm.

[0009] Further, the first prism pair includes a first prism and a second prism, and the first prism and the second prism are sequentially arranged between the first mirror and the blade; the second prism pair includes a third prism and a fourth prism, and the third prism and the fourth prism are sequentially arranged between the blade and the second mirror.

[0010] Further, the working wavelengths of the first prism, the second prism, the third prism, and the fourth prism are all 600 - 900 nm.

[0011] Further, it further includes a beam splitter and a second spectrometer;

[0012] The beam splitter is arranged between the second mirror and the resonant galvanometer scanner, and the second spectrometer is arranged on the side of the beam splitter. Among them, the beam splitter is used to split the reflection optical path of the second mirror into a reflection optical path and a transmission optical path. The reflection optical path formed by the beam splitter enters the second spectrometer, and the transmission optical path formed by the beam splitter is incident on the resonant galvanometer scanner.

[0013] Further, the focal length of the condenser lens is 15 - 30 mm, and the focal length of the lens is 30 - 100 mm.

[0014] The present invention also provides a coherent anti-Stokes Raman scattering spectroscopy measurement and microscopic imaging method, using the coherent anti-Stokes Raman scattering spectroscopy measurement and microscopic imaging device described above. Among them, the coherent anti-Stokes Raman scattering spectroscopy measurement and microscopic imaging method includes a spectroscopy measurement step;

[0015] The spectroscopy measurement step is specifically as follows:

[0016] Coarsely adjust the focus of the objective lens to focus the laser on the sample to be measured until the original coherent Raman scattering signal observed in the first spectrometer or photomultiplier tube; adjust the positions of the first mirror, the second mirror and the condenser lens so that the intensity of the coherent Raman scattering spectrum measured in the first spectrometer is maximized;

[0017] Adjust the positions of the first prism pair and the second prism pair to compensate for the dispersion of the laser on the sample to be measured until the original coherent Raman scattering signal no longer increases;

[0018] Observe the intensity of the coherent Raman scattering spectrum measured in the first spectrometer, and adjust the position of the notch filter so that the laser and the original coherent Raman scattering signal observed from the objective lens no longer increase;

[0019] Adjust the fiber optic angle of the first spectrometer until the original coherent Raman scattering signal no longer increases;

[0020] Adjust the blade to different positions to generate two different laser spectral steep-edge wavelengths, and use the first spectrometer to record the original coherent Raman scattering spectral data at the two different laser spectral steep-edge wavelengths respectively;

[0021] Obtain the measurement result of the coherent anti-Stokes Raman scattering spectrum of the sample to be measured according to the original coherent Raman scattering spectral data at the two different laser spectral steep-edge wavelengths.

[0022] Further, the process of adjusting the blade to different positions to generate two different laser spectral steep-edge wavelengths and using the first spectrometer to record the original coherent Raman scattering spectral data at the two different laser spectral steep-edge wavelengths respectively is as follows:

[0023] Adjust the position of the blade until the wavelength of the laser spectral steep edge generated by the blade is the preset first steep-edge wavelength. At this time, use the first spectrometer to record the original coherent Raman scattering spectral data to obtain the original coherent Raman scattering spectral data I at the first steep-edge wavelength 1 ;

[0024] Adjust the position of the blade until the wavelength of the laser spectral steep edge generated by the blade is the preset second steep-edge wavelength. At this time, use the first spectrometer to record the original coherent Raman scattering spectral data to obtain the original coherent Raman scattering spectral data I at the second steep-edge wavelength 2 .

[0025] Further, the preset first steep-edge wavelength is 779.3 nm, and the preset second steep-edge wavelength is 779.8 nm.

[0026] Further, the coherent anti-Stokes Raman scattering spectrum measurement and microscopic imaging method further includes a microscopic imaging step;

[0027] Among them, the microscopic imaging step is specifically as follows:

[0028] Select the reflection wavelength of the notch filter to determine the characteristic vibration mode of the sample to be measured; adjust the position of the photomultiplier tube to maximize the voltage value or current value of the measurement signal output by the photomultiplier tube;

[0029] Adjust the parameters of the resonant-galvanometer scanner, observe the amplitude and phase changes of the signal output by the lock-in amplifier, and record the measurement signal output by the photomultiplier tube;

[0030] Process the measurement signal output by the photomultiplier tube to obtain the coherent anti-Stokes Raman scattering microscopic imaging result of the sample to be measured.

[0031] Compared with the prior art, the beneficial effects of the present invention are:

[0032] The present invention provides a coherent anti-Stokes Raman scattering spectrum measurement and microscopic imaging device and method. By setting a blade between the first prism pair and the second prism pair, the laser spectrum is cropped by the shielding effect of the blade edge to generate a steep edge of the laser spectrum, and the spectral steep edge is used as a marker for the probe pulse, replacing the combined effect of the notch filter and the ultra-steep long-pass filter achieved by precision coating of quartz glass in the existing device, reducing energy consumption and cost while making the overall structure of the device simple and greatly reducing the operation difficulty of the device; secondly, using a resonant-galvanometer scanner to replace the ordinary galvanometer scanner in the traditional single-beam CARS method can increase the single-pixel imaging speed to less than 100 microseconds, which is 5 times faster than the traditional single-beam CARS microscopic imaging speed based on notch filtering, and can be better applied to the rapid imaging process in the biomedical field. Description of the Drawings

[0033] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the following will briefly introduce the drawings required for use in the description of the embodiments or the prior art. Obviously, the following drawings are only some embodiments of the present application. For those of ordinary skill in the art, other drawings can be obtained based on these drawings without creative efforts.

[0034] Figure 1 It is a schematic diagram of the overall structure of the spectrum measurement and microscopic imaging device described in the present invention;

[0035] Figure 2 It is a laser spectrum diagram before and after shaping the incident laser spectrum; among them, Figure 2 (a) is the laser spectrum before shaping the incident laser spectrum; Figure 2(b) is the incident laser spectrum when the steep-edge wavelength of the laser spectrum generated by the blade is 779.3 nm;

[0036] Figure 3 This is the chloroform original coherent anti-Stokes Raman scattering spectrum based on the blade in the present invention and the corresponding extracted chloroform Raman spectrum; Figure 3 (a) is the chloroform original coherent anti-Stokes Raman scattering spectrum measured by the blade at 779.3 nm and 779.8 nm; Figure 3 (b) is the chloroform Raman spectrum obtained by processing the original coherent anti-Stokes Raman scattering spectrum;

[0037] Figure 4 This is the microscopic imaging diagram of a rat heart slice realized based on the edge of the blade in the present invention.

[0038] Among them, 1 is a femtosecond pulsed laser, 2 is the first mirror, 3 is the first prism pair, 4 is the second prism pair, 5 is the blade, 6 is the third prism, 7 is the fourth prism, 8 is the second mirror, 9 is a resonant-galvanometer scanner, 10 is an objective lens, 11 is the sample to be measured, 12 is a condenser lens, 13 is an ultra-steep short-pass filter, 14 is a notch filter, 15 is a lens, 16 is the first spectrometer, 17 is a photomultiplier tube, 18 is a lock-in amplifier, 19 is a data acquisition card, 20 is a beam splitter, and 21 is the second spectrometer. Specific embodiments

[0039] In order to make the technical problems, technical solutions and beneficial effects solved by this application clearer and more understandable, the technical solutions in the embodiments of this application will be clearly and completely described below in conjunction with the accompanying drawings in the embodiments of this application; obviously, the described embodiments are only part of the embodiments of this application, rather than all of them. All other embodiments obtained by those of ordinary skill in the art based on the embodiments in this application without creative efforts belong to the scope of protection of this application.

[0040] As shown in the attached Figure 1 As shown in the figure, the present invention provides a coherent anti-Stokes Raman scattering spectrum measurement and microscopic imaging device, including a femtosecond pulsed laser 1, a first mirror 2, a first prism pair, a blade 3, a second prism pair, a second mirror 8, a resonant-galvanometer scanner 9, an objective lens 10, a sample to be measured 11, a condenser lens 12, an ultra-steep short-pass filter 13, a notch filter 14, a lens 15, a first spectrometer 16, a photomultiplier tube 17, a lock-in amplifier 18, a data acquisition card 19, a beam splitter 20 and a second spectrometer 21.

[0041] The femtosecond pulse laser 1 is used to emit laser pulses, and the laser pulses are ultrashort pulses; wherein, the central wavelength of the ultrashort pulses is 790 - 805 nm, the bandwidth is 10 - 70 nm, the repetition frequency is 1 - 100 MHz, and the pulse width is 5 - 100 fs; preferably, the ultrashort pulses use near-infrared band lasers, the repetition frequency is 80 MHz, and the pulse width is 10 fs, aiming to achieve CARS excitation with low average power using the high peak power of ultrasharp pulses, and having low phototoxicity to biological samples.

[0042] The laser pulses are sequentially irradiated on the sample to be measured 11 placed on the two-dimensional precision translation stage after passing through the first reflector 2, the first prism pair, the edge of the blade 5, the second prism pair, the second reflector 8, the beam splitter 20, the resonant-galvanometer scanner 9, and the objective lens 10, and form scattered signals; wherein, the objective lens 10 is installed on the first three-dimensional precision translation stage.

[0043] The first prism pair includes a first prism 3 and a second prism 4, and the first prism 3 and the second prism 4 are sequentially arranged between the first reflector 2 and the blade 5; the blade 5 is installed on a one-dimensional precision translation stage, and the blade 5 is made of stainless steel and is used to generate a steep edge of the laser spectrum by means of occlusion; preferably, the thickness of the blade 5 is 0.2 - 0.6 mm, and the length is 10 - 50 mm; the second prism pair is used for spectral shaping and dispersion compensation, and includes a third prism 6 and a fourth prism 7, and the third prism 6 and the fourth prism 7 are sequentially arranged between the blade 5 and the second reflector 8; wherein, the working wavelengths of the first prism 3, the second prism 4, the third prism 6, and the fourth prism 7 are all 600 - 900 nm.

[0044] The transmitted part of the scattered signal passes through the sample to be measured 11 and then passes through the condenser lens 12 and the ultrasharp short-pass filter 13, and then is divided into two paths by the notch filter 14; one path enters the first spectrometer 16 after passing through the lens 15 for coherent anti-Stokes Raman scattering spectroscopy measurement; the other path enters the lock-in amplifier 18 after passing through the photomultiplier tube 17 for coherent anti-Stokes Raman scattering microscopy imaging; wherein, the condenser lens 11 is arranged on the sample cell of the two-dimensional precision translation stage, and the transmissive lens 12 is arranged on the second three-dimensional precision translation stage; the focal length of the transmissive lens is 15 - 30 mm, and the focal length of the lens 15 is 30 - 100 mm; preferably, the focal length of the lens 15 is 50 mm to ensure high fiber coupling efficiency of the first spectrometer 16.

[0045] The output end of the first spectrometer 16 and the output end of the lock-in amplifier 18 are both connected to the input end of the data acquisition card 19, and the output end of the data acquisition card 19 is used to be connected to a computer; wherein, the data acquisition card 19 is used to collect the data signals of the first spectrometer 16 and the lock-in amplifier 18, and upload them to the computer for data analysis and processing.

[0046] It should be noted that the beam splitter 20 is arranged between the second mirror 8 and the resonant galvanometer scanner 9, and the second spectrometer 21 is arranged on the side of the beam splitter 20; the reflection optical path of the second mirror 8 forms a reflection optical path and a transmission optical path after passing through the beam splitter 20, the reflection optical path formed by the beam splitter 20 enters the second spectrometer 21, and the transmission optical path formed by the beam splitter 20 is incident on the resonant galvanometer scanner 9; wherein, the second spectrometer 21 is used to observe the wavelength of the laser spectral steep edge generated by the blade.

[0047] Working principle:

[0048] (1) When using the coherent anti-Stokes Raman scattering spectroscopy measurement and microscopy imaging device for spectroscopy measurement, the specific steps are as follows:

[0049] S1. Start the femtosecond pulsed laser 1, roughly adjust the focus of the objective lens 10 to focus the laser on the sample to be measured 11 until the original coherent Raman scattering signal is observed in the first spectrometer 16 or the photomultiplier tube 17; at the same time, adjust the positions of the first mirror 2, the second mirror 8 and the condenser lens 12 to make the intensity of the coherent Raman scattering spectrum measured in the first spectrometer 16 the largest; specifically, during the process of roughly adjusting the focus of the objective lens 10 to focus the laser on the sample to be measured 11, by adjusting the three-dimensional precision translation stage installed with the objective lens 10, the focus position of the objective lens 10 is adjusted, and the pitch angle and collimation of the laser are adjusted; wherein, when the laser is focused on the sample to be measured 10, the coupling efficiency between the detection element and the collected Raman scattering spectrum signal can be effectively improved.

[0050] S2. Adjust the relative positions of the first prism 3, the second prism 4, the third prism 6 and the fourth prism 7 to compensate for the dispersion of the laser on the sample to be measured 11 until the original coherent Raman scattering signal no longer increases; specifically, the first prism 3, the second prism 4, the third prism 6 and the fourth prism 7 are respectively arranged on one-dimensional precision translation stages, and the corresponding one-dimensional precision translation stages are adjusted to change the relative positions of the first prism 3, the second prism 4, the third prism 6 and the fourth prism 7, and at the same time, the change of the intensity of the original coherent Raman scattering spectrum is observed by using the first spectrometer 16 until the intensity of the original coherent Raman scattering spectrum no longer increases; wherein, the group velocity dispersion generated by the laser passing through the objective lens is compensated.

[0051] S3. Observe the intensity of the coherent Raman scattering spectrum measured in the first spectrometer 16, and adjust the position of the notch filter 14 until the original coherent Raman scattering signal from the laser to the objective lens 10 no longer increases. Specifically, observe the intensity of the coherent Raman scattering spectrum measured in the first spectrometer 16, and by adjusting the bracket that fixes the notch filter 14, make the original coherent Raman scattering spectrum signal to be observed no longer increase. At this time, the laser and the objective lens 10 are matched to the optimal position to make the incident laser pass through the objective lens 10 as much as possible.

[0052] S4. Adjust the fiber angle of the first spectrometer 16 until the original coherent Raman scattering signal no longer increases.

[0053] S5. Adjust the blade 5 to different positions to generate two different laser spectral steep-edge wavelengths, and use the first spectrometer 16 to record the original coherent Raman scattering spectrum data at the two different laser spectral steep-edge wavelengths respectively. Obtain the measurement result of the coherent anti-Stokes Raman scattering spectrum of the sample to be measured 11 according to the original coherent Raman scattering spectrum data at the two different laser spectral steep-edge wavelengths. Among them, the measurement result of the coherent anti-Stokes Raman scattering spectrum of the sample to be measured 11 is specifically:

[0054]

[0055] Among them, I Raman is the measurement result of the coherent anti-Stokes Raman scattering spectrum of the sample to be measured 11; I 1 is the original coherent Raman scattering spectrum data at the first steep-edge wavelength; I 2 is the original coherent Raman scattering spectrum data at the second steep-edge wavelength; I NRB is an intermediate variable; is the result of smooth filtering the original coherent Raman scattering spectrum data at the first steep-edge wavelength using Matlab; is the result of smooth filtering the original coherent Raman scattering spectrum data at the second steep-edge wavelength using Matlab.

[0056] Specifically, the process of adjusting the blade 5 to different positions to generate two different laser spectral steep-edge wavelengths and using the first spectrometer 16 to record the original coherent Raman scattering spectrum data at the two different laser spectral steep-edge wavelengths respectively is as follows:

[0057] S51. Use a one-dimensional precision translation stage to adjust the position of the blade 5, and use the second spectrometer 21 to observe the wavelength of the blade truncation edge until the edge wavelength is adjusted to 779 nm.

[0058] S52. Use a one-dimensional precision translation stage to adjust the position of the blade 5 until the wavelength of the laser spectral steep edge generated by the blade 5 is the preset first steep edge wavelength. At this time, use the first spectrometer 16 to record the original coherent Raman scattering spectral data, and obtain the original coherent Raman scattering spectral data I at the first steep edge wavelength. 1 ; Among them, the preset first steep edge wavelength is 779.3 nm.

[0059] S53. Use a one-dimensional precision translation stage to adjust the position of the blade 5 until the wavelength of the laser spectral steep edge generated by the blade 5 is the preset second steep edge wavelength. At this time, use the first spectrometer 16 to record the original coherent Raman scattering spectral data, and obtain the original coherent Raman scattering spectral data I at the second steep edge wavelength. 2 ; Among them, the preset second steep edge wavelength is 779.8 nm; furthermore, perform differential and normalization processing on the original coherent Raman scattering spectral data I at the first steep edge wavelength 1 and the original coherent Raman scattering spectral data I at the second steep edge wavelength 2 to obtain the coherent anti-Stokes Raman scattering spectral measurement result of the sample to be measured 11.

[0060] It should be noted that according to the coherent anti-Stokes Raman scattering spectral measurement result of the sample to be measured 11, use Matlab software to draw a graph with the vibration frequency as the horizontal axis and the CARS spectral intensity as the vertical axis to obtain the spectral intensity change graph of different vibration frequencies.

[0061] (2) When using the coherent anti-Stokes Raman scattering spectral measurement and microscopic imaging device for microscopic imaging, the specific steps are as follows:

[0062] S6. Prepare a rat heart section and place it on a glass slide, and place the glass slide loaded with the rat heart section as the sample to be measured 11 on the sample slot of the two-dimensional precision translation stage.

[0063] S7. Select the reflection wavelength of the notch filter 14 to determine the characteristic vibration mode of the sample to be measured 11; among them, the reflection wavelength of the notch filter 14 is selected as 792.5 nm; adjust the position of the photomultiplier tube 17 so that the voltage value or current value of the measurement signal output by the photomultiplier tube 17 is the largest.

[0064] S8. Adjust the parameters of the resonant galvanometer scanner 9, observe the amplitude and phase changes of the output signal of the lock-in amplifier 18, and record the measurement signal output by the photomultiplier tube 17. Specifically, when the relative distance between the objective lens 10 and the sample to be measured 11 remains unchanged, establish an xoy coordinate system with the horizontal plane where the sample to be measured 11 is located as the coordinate plane, and determine the coordinates of the position of the light spot on the sample to be measured 11. When the y value of the coordinate of the position of the light spot on the sample to be measured 11 is a constant value, increase the voltage value applied to the x-axis direction of the two-dimensional precision translation stage where the sample to be measured 11 is located, and the x value of the light spot coordinate is translated in steps of 1 μm. At the same time, record the signals of the photomultiplier tube at 120 different x values. When the y value of the coordinate of the position of the light spot on the sample to be measured 11 is y = y + 1 μm, record the signals of the photomultiplier tube 17 at 120 different x values again. When y = y + 120 μm, record the signals of the photomultiplier tube 17 at 120 different x values again, scan an area of 120 μm × 120 μm, and record all the values on the 120 × 120 two-dimensional matrix grid points.

[0065] S9. Process the measurement signal output by the photomultiplier tube 17 to obtain the coherent anti-Stokes Raman scattering microscopic imaging result of the sample to be measured 11. Specifically, use Labview software and Matlab software to process the measurement signal output by the photomultiplier tube 17 to obtain the coherent anti-Stokes Raman scattering microscopic imaging result of the sample to be measured 11.

[0066] In the present invention, by setting a blade between two prism pairs and making the pulsed laser pass through the edge of the blade, the detection light is marked by using the shielding effect of the blade to generate a steep edge of the laser spectrum, so as to replace the combined effect of the notch filter and the ultra-steep long-pass filter realized by precision coating of quartz glass, reducing energy consumption and cost at the same time. Secondly, by introducing a resonant galvanometer scanner and using the resonant galvanometer scanner to replace the ordinary galvanometer scanner in the traditional single-beam CARS scheme, the single-pixel imaging speed is increased to less than 100 microseconds, realizing faster terahertz-band CARS microscopic imaging.

[0067] Specific experimental description:

[0068] Taking the experimental process of spectral measurement of chloroform and microscopic imaging of rat heart slices as an example, the spectral measurement and microscopic imaging device of the present invention will be described in detail.

[0069] A femtosecond pulse laser 1 is used to generate a laser pulse with a pulse width of 15 fs, a central wavelength of 795 nm, and a bandwidth of 30 nm. The laser pulse first passes through the first mirror 2, and then successively passes through the first prism 3, the second prism 4, the edge of the blade 5, the third prism 6, and the fourth prism 7 for spectral shaping and dispersion compensation, and then passes through the second mirror 8. Then it passes through the resonant-galvanometer scanner 9, and then is focused on the sample to be measured 11 by the objective lens 10 placed on the first three-dimensional precision translation stage. Among them, the sample to be measured 11 is placed in the sample groove of a two-dimensional precision translation stage. The transmitted signal is converged by the condenser lens 12 placed on the second three-dimensional precision translation stage. The incident laser is filtered out by the ultra-steep short-pass filter 13 in the transmitted part. Among them, the transmitted light is introduced into the optical fiber connected to the first spectrometer 16 through a 50-mm lens 15 for measuring the original coherent Raman scattering spectrum. The reflected light is selectively reflected through the notch filter 14 and introduced into the photomultiplier tube 17, and enters the lock-in amplifier 18 for Raman scattering microscopy imaging. The electrical signals converted by the first spectrometer 16 and the photomultiplier tube 17 are extracted by the data acquisition card 19 and connected to a computer for data processing. Among them, by measuring the laser spectrum shaped by the blade, the light passing through the first mirror is split into two beams by the beam splitter 20, and the first reflected beam passes through the second spectrometer 21 for detecting the steep edge position generated by the blade 5.

[0070] Model descriptions of the above components:

[0071] The model of the femtosecond pulsed laser 1 is: Thorlabs, OCTAVIUS-85M-HP; the model of the first reflector 2 is: Thorlabs, UM10-AG-10; the models of the first prism 3, the second prism 4, the third prism 6 and the fourth prism 7 are all AFS-FS; the model of the blade 5 is Personna GEM, 0.23 mm thick; the model of the second reflector 8 is: Thorlabs, UM10-AG-10; the model of the objective lens 10 is: Newport, 20x, 0.4NA; the model of the condenser lens 12 is: Edmund Optics, 0.5NA; the model of the ultra-steep short-pass filter 13 is: Semrock SP01-785RU; the model of the notch filter 14 is: Unaxis Blazers; the model of the lens 15 is: Thorlabs, AC254-030-AB; the model of the first spectrometer 16 is: Jobin Yvon Triax 320; the models of the beam splitters 20 are all: Thorlabs, ME2-M01; the model of the second spectrometer 21 is all CCS175; the model of the one-dimensional precision translation stage is: Thorlabs, PT1B; the model of the two-dimensional precision translation stage is: Mad City Labs, Inc. Nano-Bios; the model of the three-dimensional precision translation stage is: Thorlabs, RB13M / M.

[0072] When chloroform is used as the sample 11 to be measured for spectral measurement, start the laser and all spectrometers, and observe the incident laser spectrum and the position of the sharp edge of the blade, as shown in the appendix Figure 2 shown; among them, the appendix Figure 2 (a) is the laser spectrum before the shaping of the incident laser spectrum; the appendix Figure 2 (b) is the incident laser spectrum when the wavelength of the sharp edge of the laser spectrum generated by the blade is 779.3 nm; by measuring the original CARS spectrum of the blade at 779.3 nm, and then recording the original CARS spectrum of the blade at 779.8 nm; then extract the chloroform Raman spectrum according to the original CARS spectrum of the blade at 779.3 nm and the original CARS spectrum of chloroform at 779.8 nm of the blade, as shown in the appendix Figure 3 shown; among them, the appendix Figure 3 (a) is the CARS spectrum of chloroform measured by the blade at 779.3 nm and 779.8 nm; the appendix Figure 3 (b) is the chloroform Raman spectrum obtained by processing the original CARS spectrum.

[0073] When CARS microscopic imaging is performed on a rat heart slice as the sample 11 to be tested, the reflection wavelength of the notch filter 14 is selected to meet the Raman scattering microscopic imaging of different vibration frequencies of the sample; when the reflection wavelength of the notch filter 14 is 792.5nm, microscopic imaging of the rat heart slice is performed, as shown in the attached figure. Figure 4 shown.

[0074] The spectrum measurement and microscopic imaging device of the present invention utilizes a resonance-galvanometer scanner to perform high-frequency modulation of an incident laser at a frequency of more than 10 kHz, thereby performing high-frequency modulation of a resonance term in a Raman scattering signal; since the resonant Raman scattering in the generated Raman scattering signal is only related to the position of a blade steep edge, the non-resonant Raman scattering is independent of the blade position and can be used as a local oscillator; the non-resonant signal is removed by phase-locked amplification, and a weaker resonant signal in the Raman scattering signal is extracted; by slightly adjusting the position of the steep edge, two groups of original Raman scattering spectra are measured, and the two are differentiated and normalized to obtain a low-frequency vibration spectrum or a Raman spectrum, including a low-frequency band vibration spectrum; by introducing an ultra-steep short-pass filter, it is ensured that the incident laser is infinitely close to the detected Raman scattering signal, thereby realizing the detection of the low-frequency band vibration spectrum.

[0075] In the present invention, when the thickness of the blade is set to 0.2 mm, a high spectral resolution can be guaranteed; since biomacromolecules, such as proteins and DNA, have a large-amplitude vibration mode in a low-frequency band, a CARS system with a high spectral resolution and fast microscopic imaging are realized by using a blade and a resonance-galvanometer scanner; since the blade is thin enough, the trimmed spectral edge is steep enough, and the power consumed by the laser when generating the steep edge is less than half of the power consumed by the notch filter and the ultra-steep filter together, the energy consumption is low; therefore, the common function of the notch filter and the ultra-steep filter can be realized by using one blade, so that the system is greatly simplified; the blade production only requires simple grinding, and does not require complex processes such as precision coating, so the cost is low; the steep edge generated by the blade on the laser spectrum only requires the blade to be fixed on a one-dimensional translation stage, and does not require precise rotation, thus eliminating the need for a precise rotation stage, so the operation is relatively simple.

[0076] In the present invention, a prism is used to expand a femtosecond pulse laser in space, and the sharp edge of a blade is used to achieve laser spectrum clipping, and the steep edge is used as a marker for detecting pulses, thereby reducing costs and making the system more compact and lightweight; secondly, a resonant-galvanometer scanner is used to replace the ordinary galvanometer scanner in the traditional single-beam CARS solution, so that the single-pixel imaging speed is increased to less than 100 microseconds, which is 5 times faster than the traditional single-beam CARS microscopic imaging speed based on notch filtering, and can be applied to rapid imaging in the biomedical field.

[0077] The above embodiments are merely one of the implementation manners capable of implementing the technical solution of the present invention. The scope of protection required by the present invention is not limited solely by this embodiment, but also includes any changes, substitutions, and other implementation manners that are easily conceivable by any person skilled in the art within the technical scope disclosed by the present invention.

Claims

1. A coherent anti-Stokes Raman scattering spectrum measurement and microscopic imaging device, characterized in that: The invention comprises a femtosecond pulse laser (1), wherein the laser pulse generated by the femtosecond pulse laser (1) is sequentially irradiated onto a sample to be tested (11) placed on a two-dimensional precision translation stage after passing through a first reflector (2), a first prism pair, an edge of a blade (5), a second prism pair, a second reflector (8), a resonance-galvanometer scanner (9) and an objective lens (10), thereby forming a scattering signal; wherein the blade (5) is used to generate a steep edge of a laser spectrum by shielding; The scattered signal passes through the transmission part of the sample to be measured (11), a condenser (12), an ultra-steep short-pass filter (13), and then a notch filter (14) and is divided into two paths; one path passes through a lens (15) and enters a first spectrometer (16) for coherent anti-Stokes Raman scattering spectrum measurement; the other path passes through a photomultiplier tube (17) and enters a phase-locked amplifier (18) for coherent anti-Stokes Raman scattering microscopic imaging; The thickness of the blade (5) is 0.2-0.6 mm, and the length of the blade (5) is 10-50 mm; The first prism pair comprises a first prism (3) and a second prism (4), the first prism (3) and the second prism (4) being arranged in sequence between the first reflector (2) and the blade (5); the second prism pair comprises a third prism (6) and a fourth prism (7), the third prism (6) and the fourth prism (7) being arranged in sequence between the blade (5) and the second reflector (8).

2. A coherent anti-Stokes Raman scattering spectrum measurement and microscopic imaging device according to claim 1, characterized in that: The operating wavelengths of the first prism (3), the second prism (4), the third prism (6) and the fourth prism (7) are all 600-900 nm.

3. The coherent anti-Stokes Raman scattering spectrum measurement and microscopic imaging device according to claim 1, characterized in that: It also includes a beam splitter (20) and a second spectrometer (21); The beam splitter (20) is arranged between the second reflector (8) and the resonance-galvanometer scanner (9), and the second spectrometer (21) is arranged on the side of the beam splitter (20); wherein the beam splitter (20) is used to split the reflected light path of the second reflector (8) into a reflected light path and a transmitted light path, the reflected light path formed by the beam splitter (20) enters the second spectrometer (21), and the transmitted light path formed by the beam splitter (20) enters the resonance-galvanometer scanner (9).

4. The coherent anti-Stokes Raman scattering spectrum measurement and microscopic imaging device according to claim 1, characterized in that: The focal length of the condenser (12) is 15-30 mm, and the focal length of the lens (15) is 30-100 mm.

5. A coherent anti-Stokes Raman scattering spectrum measurement and microscopic imaging method, characterized in that: Using the coherent anti-Stokes Raman scattering spectrum measurement and microscopic imaging device as described in any one of claims 1 to 4; wherein the coherent anti-Stokes Raman scattering spectrum measurement and microscopic imaging method comprises a spectrum measurement step; The spectrum measurement steps are as follows: Coarsely adjusting the focus of the objective lens (10) so that the laser is focused on the sample to be measured (11) until the original coherent Raman scattering signal is observed in the first spectrometer (16) or the photomultiplier tube (17); adjusting the positions of the first reflector (2), the second reflector (8) and the condenser (12) so that the intensity of the coherent Raman scattering spectrum measured in the first spectrometer (16) is maximized; Adjusting the positions of the first prism pair and the second prism pair so that the dispersion of the laser light on the sample (11) to be measured is compensated until the original coherent Raman scattering signal no longer increases; Observing the intensity of the coherent Raman scattering spectrum measured in the first spectrometer (16), and adjusting the position of the notch filter (14) so ​​that the laser and the objective lens (10) are aligned until the observed original coherent Raman scattering signal no longer increases; Adjusting the optical fiber angle of the first spectrometer (16) until the original coherent Raman scattering signal no longer increases; Adjusting the blade (5) to different positions to generate two different laser spectrum steep edge wavelengths, and using a first spectrometer (16) to respectively record original coherent Raman scattering spectrum data at the two different laser spectrum steep edge wavelengths; The coherent anti-Stokes Raman scattering spectrum measurement results of the sample (11) to be tested are obtained based on the original coherent Raman scattering spectrum data at two different laser spectrum steep edge wavelengths.

6. A coherent anti-Stokes Raman scattering spectrum measurement and microscopic imaging method according to claim 5, characterized in that: The process of adjusting the blade (5) to different positions to generate two different laser spectrum steep edge wavelengths, and using the first spectrometer (16) to respectively record the original coherent Raman scattering spectrum data at the two different laser spectrum steep edge wavelengths is specifically as follows: The position of the blade (5) is adjusted until the wavelength of the steep edge of the laser spectrum generated by the blade (5) is a preset first steep edge wavelength, and at this time, the original coherent Raman scattering spectrum data is recorded using the first spectrometer (16) to obtain the original coherent Raman scattering spectrum data at the first steep edge wavelength. ; The position of the blade (5) is adjusted until the wavelength of the steep edge of the laser spectrum generated by the blade (5) is the preset second steep edge wavelength, and at this time, the original coherent Raman scattering spectrum data is recorded using the first spectrometer (16) to obtain the original coherent Raman scattering spectrum data at the second steep edge wavelength. .

7. A coherent anti-Stokes Raman scattering spectrum measurement and microscopic imaging method according to claim 6, characterized in that: The preset first steep edge wavelength is 779.3 nm, and the preset second steep edge wavelength is 779.8 nm.

8. A coherent anti-Stokes Raman scattering spectrum measurement and microscopic imaging method according to claim 5, characterized in that: The coherent anti-Stokes Raman scattering spectrum measurement and microscopic imaging method further comprises a microscopic imaging step; Wherein, the microscopic imaging step is specifically as follows: Selecting the reflection wavelength of the notch filter (14) to determine the characteristic vibration mode of the sample (11) to be measured; adjusting the position of the photomultiplier tube (17) so that the voltage value or current value of the measurement signal output by the photomultiplier tube (17) is maximized; Adjusting the parameters of the resonance-galvanometer scanner (9), observing the amplitude and phase changes of the output signal of the lock-in amplifier (18), and recording the measurement signal output by the photomultiplier tube (17); The measurement signal output by the photomultiplier tube (17) is processed to obtain a coherent anti-Stokes Raman scattering microscopic imaging result of the sample (11) to be measured.

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