A CARS Spectroscopy Measurement and Microscopy Imaging Device and Method

Abstract

The present invention belongs to the technical field of Raman scattering microscopy imaging, and discloses a CARS spectrum measurement and microscopy imaging device and method, which includes a femtosecond pulsed laser. The laser pulse generated by the femtosecond pulsed laser is sequentially irradiated on a sample to be measured after passing through a dichroic mirror, a grating, a curved mirror, a first needle tip reflector, a second reflector, a third reflector, an ultra-steep long-pass filter, a resonant galvanometer scanner and an objective lens, and a scattering signal is formed. The transmitted part of the scattering signal passing through the sample to be measured sequentially passes through a condenser lens and an ultra-steep short-pass filter, and then is split into two paths by a first beam splitter. One path enters a first spectrometer through a lens for CARS spectrum measurement; the other path enters a lock-in amplifier through a notch filter and a photomultiplier tube for CARS microscopy imaging. Among them, the needle tip is arranged on a one-dimensional translation stage for notch shaping of the laser spectrum. The present invention generates a laser spectrum notch mark through the needle tip, improving the spectral resolution.

Description

Technical Field

[0001] The present invention belongs to the technical field of Raman scattering microscopy, and particularly relates to a CARS spectroscopy measurement and microscopy device and method. Background Art

[0002] Coherent anti-Stokes Raman scattering (CARS) is an optical process of four-wave mixing (FWM) involving the interaction of a pump light, a Stokes light, an anti-Stokes light, and sample molecules. It belongs to a nonlinear optical effect based on third-order nonlinear Raman scattering, and its signal intensity is 5 - 6 orders of magnitude higher than that of spontaneous Raman scattering (SPRS). In the biomedical field, especially in live cell imaging, it has been widely used.

[0003] CARS devices generally adopt multi-beam or multi-light source schemes to meet the frequency component requirements of pump photons, Stokes photons, and detection photons, and all excitation beams must spatially coincide; to simplify traditional CARS devices, some researchers have used notch filters to achieve single-beam CARS; however, compared with traditional multi-beam CARS, due to the introduction of notch filters, the resolution of the CARS device is lower, the structure is complex, and the equipment price is expensive; secondly, since traditional CARS devices are usually equipped with galvanometer scanners, and the modulation speed of galvanometer scanners is limited, the pixel imaging speed is often limited to the millisecond level. Summary of the Invention

[0004] Aiming at the technical problems existing in the prior art, the present invention provides a CARS spectroscopy measurement and microscopy device and method to solve the technical problems of the existing CARS device with a notch filter having lower resolution, complex structure, and high price.

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

[0006] The present invention provides a CARS spectroscopy measurement and microscopy device, including a femtosecond pulsed laser. The laser pulse generated by the femtosecond pulsed laser is sequentially irradiated on a sample to be measured placed on a two-dimensional precision translation stage through a dichroic mirror, a grating, a curved mirror, a first needle mirror, a second mirror, a third mirror, an ultra-steep long-pass filter, a resonant-galvanometer scanner, and an objective lens, and a scattering signal is formed;

[0007] The transmitted part of the scattering signal passing through the sample to be measured is sequentially passed through a condenser lens and an ultra-steep short-pass filter, and then split into two paths by a first beam splitter; one path enters a first spectrometer through a lens for CARS spectroscopy measurement; the other path enters a lock-in amplifier through a notch filter and a photomultiplier tube for CARS microscopy;

[0008] Among them, the tip is disposed on a one-dimensional translation stage for notch shaping of the laser spectrum.

[0009] Furthermore, the tip adopts a steel tip structure; wherein, the diameter of the tip is 0.1 - 0.5 mm, and the length is 1 - 20 mm.

[0010] Furthermore, it further includes a second beam splitter and a second spectrometer;

[0011] The second beam splitter is disposed between the third mirror and the ultra-steep long-pass filter, and the second spectrometer is disposed on the side of the second beam splitter;

[0012] Among them, the reflected light path of the third mirror forms a reflected light path and a transmitted light path after passing through the second beam splitter. The reflected light path formed by the second beam splitter enters the second spectrometer, and the transmitted light path formed by the second beam splitter is incident on the ultra-steep long-pass filter.

[0013] Furthermore, it further includes a third beam splitter and a third spectrometer;

[0014] The third beam splitter is disposed between the ultra-steep long-pass filter and the resonant-galvanometer scanner, and the third spectrometer is disposed on the side of the third beam splitter;

[0015] Among them, the transmitted light of the ultra-steep long-pass filter forms a reflected light path and a transmitted light path after passing through the third beam splitter. The reflected light path formed by the third beam splitter enters the third spectrometer, and the transmitted light path formed by the third beam splitter is incident on the resonant-galvanometer scanner.

[0016] Furthermore, the blazing wavelength of the grating is 750 nm; wherein, there are 1200 grooves per millimeter.

[0017] The present invention also provides a CARS spectrum measurement and microscopic imaging method. Using the CARS spectrum measurement and microscopic imaging device, the CARS spectrum measurement and microscopic imaging method includes a CARS spectrum measurement step;

[0018] Among them, the CARS spectrum measurement step is specifically as follows:

[0019] Coarsely adjust the focus of the objective lens to focus the laser on the sample to be measured until the original CARS signal is observed in the first spectrometer; adjust the positions of the first mirror, the second mirror, the third mirror and the condenser lens to maximize the CARS spectrum intensity measured in the first spectrometer;

[0020] Adjust the relative positions of the curved mirror and the grating to compensate for the dispersion of the laser on the sample to be measured until the original CARS signal no longer increases;

[0021] Observe the CARS spectral intensity measured in the first spectrometer, and adjust the position of the first beam splitter until the original CARS signal no longer increases;

[0022] Adjust the fiber optic angle of the first spectrometer until the original CARS signal no longer increases;

[0023] Adjust the tip to different positions to generate notches of two different wavelength sizes, and use the first spectrometer to record the original CARS spectral data under the notches of the two different wavelength sizes respectively; Obtain the CARS spectral measurement result of the sample to be measured according to the original CARS spectral data under the notches of the two different wavelength sizes.

[0024] Further, the grating is installed on a one-dimensional precision translation stage;

[0025] Among them, the process of adjusting the relative position between the curved mirror and the grating is specifically as follows:

[0026] Adjust the one-dimensional precision translation stage to change the position of the grating, thereby realizing the adjustment of the relative position between the curved mirror and the grating.

[0027] Further, the process of adjusting the tip to different positions to generate notches of two different wavelength sizes and using the first spectrometer to record the original CARS spectral data under the notches of the two different wavelength sizes respectively is specifically as follows:

[0028] Adjust the angle of the ultra-steep long-pass filter to adjust the cut-off edge wavelength of the ultra-steep long-pass filter to a preset edge wavelength;

[0029] Use the one-dimensional translation stage to adjust the position of the tip until the wavelength of the notch generated by the tip is the preset first notch wavelength; Use the first spectrometer to record the original CARS spectral data to obtain the original CARS spectral data under the first wavelength notch ;

[0030] Use the one-dimensional translation stage to adjust the position of the tip again until the wavelength of the notch generated by the tip is the preset second notch wavelength; Use the first spectrometer to record the original CARS spectral data to obtain the original CARS spectral data under the second wavelength notch 。

[0031] Further, the preset edge wavelength is 779 nm, the preset first notch wavelength is 779.6 nm, and the preset second notch wavelength is 779.9 nm.

[0032] Further, the CARS spectral measurement and microscopic imaging method further includes a microscopic imaging step;

[0033] Among them, the microscopic imaging step is specifically as follows:

[0034] 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 so that the voltage value or current value of the electrical signal output by the photomultiplier tube is maximized;

[0035] 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 electrical signal output by the photomultiplier tube;

[0036] Process the electrical signal output by the photomultiplier tube to obtain the CARS microscopic imaging result of the sample to be measured.

[0037] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0038] The present invention provides a CARS spectrum measurement and microscopic imaging device and method. By setting a tip between the curved mirror and the reflecting mirror to replace the notch filter realized by precision coating of quartz glass, the curved mirror is used to spatially expand the laser pulse generated by the femtosecond pulse laser, and a laser spectrum notch mark is generated through the shielding effect of the tip to achieve the notch shaping effect of the laser spectrum, effectively improving the spectral resolution of the device; secondly, it can reduce the power loss of the laser when generating the notch, greatly reducing the energy consumption of the device; the tip structure is simple, the manufacturing cost is low, and it does not need to be equipped with a precision rotary table for use, effectively reducing the structural complexity of the device; in addition, using a resonant-galvanometer scanner to replace the traditional galvanometer scanner can effectively improve the imaging speed and meet the fast imaging requirements in the biomedical field. Description of the Drawings

[0039] 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.

[0040] Figure 1 It is a schematic structural diagram of the CARS spectrum measurement and microscopic imaging device described in the present invention;

[0041] Figure 2 It is a diagram of the incident laser spectrum shaping results by the tip, the ultra-steep filter, and both of them in the present invention; among them, Figure 2 (a) is the incident laser spectrum measured by the second spectrometer when the tip is at 779.9 nm; Figure 2 (b) is the incident laser spectrum measured by the third spectrometer when the tip is at 775 nm; Figure 2 (c) is the incident laser spectrum measured by the third spectrometer when the tip is at 779.9 nm.

[0042] Figure 3 The original CARS spectrum of tetrabromoethane based on the tip in the present invention and the corresponding Raman spectrum of the extracted tetrabromoethane; wherein, Figure 3 (a) is the original CARS spectrum of tetrabromoethane measured by the third spectrometer when the tip is at 779.6 nm and 779.9 nm; Figure 3 (b) is the Raman spectrum of tetrabromoethane obtained by processing the original CARS spectrum.

[0043] Figure 4 The CARS microscopic imaging diagram of the rat tail tendon realized based on the tip in the present invention.

[0044] Among them, 1 is a femtosecond pulsed laser, 2 is a dichroic mirror, 3 is a grating, 4 is a curved mirror, 5 is a tip, 6 is a first reflector, 7 is a second reflector, 8 is a third reflector, 9 is an ultra-steep long-pass filter, 10 is a resonant-galvanometer scanner, 11 is an objective lens, 12 is a sample to be measured, 13 is a condenser lens, 14 is an ultra-steep short-pass filter, 15 is a first beam splitter, 16 is a lens, 17 is a first spectrometer, 18 is a notch filter, 19 is a photomultiplier tube, 20 is a lock-in amplifier, 21 is a data acquisition card, 22 is a second beam splitter, 23 is a second spectrometer, 24 is a third beam splitter, and 25 is a third spectrometer. Detailed implementation manners

[0045] In order to make the technical problems, technical solutions and beneficial effects solved by the present application clearer and more understandable, the technical solutions in the embodiments of the present application will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present application; obviously, the described embodiments are only a part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those of ordinary skill in the art without creative efforts belong to the scope of protection of the present application.

[0046] As shown in the Figure 1 accompanying drawings, the present invention provides a CARS spectrum measurement and microscopic imaging device, including a femtosecond pulsed laser 1, a dichroic mirror 2, a grating 3, a curved mirror 4, a tip 5, a first reflector 6, a second reflector 7, a third reflector 8, an ultra-steep long-pass filter 9, a resonant-galvanometer scanner 10, an objective lens 11, a sample to be measured 12, a condenser lens 13, an ultra-steep short-pass filter 14, a first beam splitter 15, a lens 16, a first spectrometer 17, a notch filter 18, a photomultiplier tube 19, a lock-in amplifier 20, a data acquisition card 21, a data processing module, a second beam splitter 22, a second spectrometer 23, a third beam splitter 24 and a third spectrometer 25.

[0047] The femtosecond pulse laser 1 is used to emit laser pulses, and the laser pulses are ultrashort laser pulses; among them, the central wavelength of the ultrashort laser 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.

[0048] The dichroic mirror 2, the grating 3, the curved mirror 4, and the tip 5 are sequentially arranged on the optical path of the laser pulse. The grating 3 is installed on a one-dimensional precision translation stage, and the curved mirror 4 is used to expand the laser pulse in space; among them, by adjusting the relative positions of the grating 3 and the curved mirror 4, dispersion compensation for the laser pulse is achieved, and a compensated laser pulse is formed; the tip 5 is arranged on a one-dimensional translation stage and is used for notch shaping of the laser spectrum, that is, notch marking is performed on the compensated laser pulse to form a laser pulse after notch marking; among them, the blaze wavelength of the grating 3 is 750 nm, and there are 1200 grooves per millimeter; the tip 5 adopts a steel tip structure; among them, the diameter of the tip 5 is 0.1 - 0.5 mm, and the length is 1 - 20 mm.

[0049] The first reflector 6 is arranged on the optical path of the laser pulse after notch marking and is used to reflect the laser pulse after notch marking to form a first reflected light beam; the first reflected light beam is incident on the second reflector 7 after passing through the curved mirror 4, the grating 3, and the dichroic mirror 2 in sequence, and a second reflected light beam is formed after being reflected by the second reflector 7.

[0050] The third reflector 8 is arranged on the optical path of the second reflected light beam, and the second reflected light beam forms a third reflected light beam after being reflected by the third reflector 8; the second beam splitter 22 is arranged on the optical path of the third reflected light beam, and the third reflected light beam forms a first reflected laser and a first transmitted laser after passing through the second beam splitter 22; the second spectrometer 23 is arranged on the optical path of the first reflected laser, and the second spectrometer 23 is used to detect the notch position generated by the tip 5; the ultra-steep long-pass filter 9 is arranged on the optical path of the first transmitted laser and is used to perform filtering processing on the first transmitted laser to obtain a first filtered light beam; the third beam splitter 24 is arranged on the optical path of the first filtered light beam, and the first filtered light beam forms a second reflected laser and a second transmitted laser after passing through the third beam splitter 24; the third spectrometer 25 is arranged on the optical path of the second reflected laser, and the third spectrometer 25 is used to detect the ultra-steep edge position generated by the ultra-steep long-pass filter 9.

[0051] The resonance-galvanometer scanner 10, the objective lens 11, the sample to be measured 12, the condenser lens 13, and the ultra-steep short-pass filter 14 are sequentially arranged on the optical path of the second transmitted laser; wherein, the objective lens 11 is mounted on a three-dimensional precision translation stage, and the sample to be measured 12 is placed on the sample slot of a two-dimensional precision translation stage; specifically, the second transmitted laser passes through the resonance-galvanometer scanner 10 and the objective lens 11 in sequence and then is focused on the sample to be measured 12, and after passing through the sample to be measured 12, a scattered signal is formed; the scattered signal is converged by the condenser lens 13 to form a converging light beam; after the converging light beam passes through the ultra-steep short-pass filter 14, the incident laser in the converging light beam is filtered out to form a transmitted light beam; wherein, the focal length of the condenser lens 13 is 15 - 30 mm.

[0052] The first beam splitter 15 is arranged on the optical path of the transmitted light beam, and the transmitted light beam forms a third reflected laser and a third transmitted laser after passing through the first beam splitter 15; the third transmitted laser is guided into the first spectrometer 17 after passing through the lens 16; the third reflected laser enters the lock-in amplifier 20 after passing through the notch filter 18 and the photomultiplier tube 19; the output end of the first spectrometer 17 and the output end of the photomultiplier tube 19 are both connected to the input end of the data acquisition card 21, and the output end of the data acquisition card 21 is connected to the input end of the data analysis module; wherein, the focal length of the lens 16 is 30 - 100 mm; preferably, the focal length of the lens 16 is 50 mm, which can effectively improve the coupling efficiency between the lens 16 and the optical fiber of the first spectrometer 17.

[0053] In the CARS spectral measurement and microscopic imaging device of the present invention, the laser pulse generated by the femtosecond pulsed laser 1 sequentially passes through the dichroic mirror 2, the grating 3, the curved mirror 4, the tip 5, the first mirror 6, the second mirror 7, the third mirror 8, the ultra-steep long-pass filter 9, the resonance-galvanometer scanner 10, and the objective lens 11, and then irradiates on the sample to be measured 12 placed on the two-dimensional precision translation stage, and a scattered signal is formed; the transmitted part of the scattered signal passes through the condenser lens 13 and the ultra-steep short-pass filter 14 in sequence after passing through the sample to be measured 12, and then is split into two paths by the first beam splitter 15; one path enters the first spectrometer 17 through the lens 16 for CARS spectral measurement; the other path enters the lock-in amplifier 20 through the notch filter 18 and the photomultiplier tube 19 for CARS microscopic imaging.

[0054] In the present invention, the reflected light path of the third mirror 8 forms a reflected light path and a transmitted light path after passing through the second beam splitter 22. The reflected light path formed by the second beam splitter 22 enters the second spectrometer 23, and the transmitted light path formed by the second beam splitter 22 is incident on the ultra-steep long-pass filter 9. The transmitted light of the ultra-steep long-pass filter 9 forms a reflected light path and a transmitted light path after passing through the third beam splitter 24. The reflected light path formed by the third beam splitter 24 enters the third spectrometer 25, and the transmitted light path formed by the third beam splitter 24 is incident on the resonant-galvanometer scanner 10.

[0055] Working principle:

[0056] (1) When using the CARS spectral measurement and microscopic imaging device of the present invention for CARS spectral measurement, the specific steps are as follows:

[0057] S1. Start the femtosecond pulsed laser 1, and roughly adjust the focus of the objective lens 11 to focus the laser on the sample to be measured 12 until the original CARS signal is observed in the first spectrometer 17 or the photomultiplier tube 19. At the same time, adjust the positions of the first mirror 6, the second mirror 7, the third mirror 8, and the condenser lens 13 to maximize the CARS spectral intensity measured in the first spectrometer 17.

[0058] Among them, the process of roughly adjusting the focus of the objective lens 11 to focus the laser on the sample to be measured 12 is specifically as follows: By adjusting the three-dimensional precision translation stage installed with the objective lens 11, the focus position of the objective lens 11 is adjusted, and the pitch angle and collimation of the laser pulse are adjusted. When the laser is focused on the sample to be measured 12, the coupling efficiency between the detection element and the collected CARS signal can be effectively improved.

[0059] S2. Adjust the relative positions of the curved mirror 4 and the grating 3 to compensate for the dispersion of the laser on the sample to be measured 12. At the same time, use the first spectrometer 17 to observe the change in the spectral intensity of the original CARS signal until the spectral intensity of the original CARS signal no longer increases. Specifically, adjust the one-dimensional precision translation stage installed with the grating 3 to change the position of the grating 3, thereby realizing the adjustment of the relative positions of the curved mirror 4 and the grating 3. By compensating for the dispersion of the laser on the sample to be measured 12, the group velocity dispersion generated by the objective lens 11 can be compensated.

[0060] S3. Observe the CARS spectral intensity measured in the first spectrometer 17, and adjust the position of the first beam splitter 15 until the original CARS signal no longer increases. At this time, the incident laser is matched to the best position with the objective lens 11, so that the incident laser can pass through the objective lens 11 as much as possible. Among them, when adjusting the first beam splitter 15, it is realized by adjusting the fixed bracket installed with the first beam splitter 15.

[0061] S4. Adjust the optical fiber angle of the first spectrometer 17 until the original CARS signal no longer increases, so as to maximize the optical coupling efficiency.

[0062] S5. Adjust the tip 5 to different positions to produce notches of two different wavelength sizes; use the first spectrometer 17 to record the original CARS spectral data under the notches of the two different wavelength sizes respectively, and obtain the CARS spectral measurement results of the sample to be measured 12 according to the original CARS spectral data under the notches of the two different wavelength sizes.

[0063] Specifically, the process of adjusting the tip 5 to different positions to produce notches of two different wavelength sizes is as follows:

[0064] S501. Adjust the angle of the ultra-steep long-pass filter 9, and use the second spectrometer 23 to observe the wavelength of the truncation edge of the ultra-steep long-pass filter, so as to adjust the truncation edge wavelength of the ultra-steep long-pass filter to a preset edge wavelength; preferably, the preset edge wavelength is 779 nm.

[0065] S502. Use the one-dimensional translation stage to adjust the position of the tip 5 until the wavelength at which the tip 5 produces a notch is the preset first notch wavelength; preferably, the preset first notch wavelength is 779.6 nm; use the first spectrometer 17 to record the original CARS spectral data to obtain the original CARS spectral data under the first wavelength notch. 。

[0066] S503. Use the one-dimensional translation stage to adjust the position of the tip 5 again until the wavelength at which the tip 5 produces a notch is the preset second notch wavelength; preferably, the preset second notch wavelength is 779.9 nm; use the first spectrometer 17 to record the original CARS spectral data to obtain the original CARS spectral data under the second wavelength notch. 。

[0067] S6. Use the data acquisition card 21 to obtain the original CARS spectral data under the first wavelength notch and the original CARS spectral data under the second wavelength notch , and upload them to the data analysis module after digital conversion; in the data analysis module, perform differentiation and normalization processing on the original CARS spectral data under the first wavelength notch and the original CARS spectral data under the second wavelength notch to obtain the CARS spectral measurement results of the sample to be measured 12; according to the CARS spectral measurement results of the sample to be measured 12, 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 CARS spectral intensity change graph of different vibration frequencies.

[0068] Among them, the CARS spectrum measurement results of the sample 12 to be measured are specifically as follows:

[0069]

[0070]

[0071] Among them, is the CARS spectrum measurement result of the sample 12 to be measured; is the original CARS spectrum data under the preset first wavelength notch; is the original CARS spectrum data under the second wavelength notch; is an intermediate variable; is the result of smooth filtering the original CARS spectrum data under the first wavelength notch using Matlab; is the result of smooth filtering the original CARS spectrum data under the second wavelength notch using Matlab.

[0072] (2) When performing CARS microscopy using the CARS spectrum measurement and microscopy imaging device of the present invention, the specific steps are as follows:

[0073] S7. Obtain an animal tissue section, place the animal tissue section on a glass slide, and use the glass slide loaded with the animal tissue section as the sample 12 to be measured, and place it on a two-dimensional precision translation stage.

[0074] S8. Use the notch filter 18 to determine the characteristic vibration mode of the sample 12 to be measured; specifically, select the reflection wavelength of the notch filter 18 to determine the characteristic vibration mode of the sample 12 to be measured; preferably, select the reflection wavelength of the notch filter 18 to be 784.6 nm; adjust the position of the photomultiplier tube 19 so that the voltage value or current value of the electrical signal output by the photomultiplier tube 19 is the largest.

[0075] S9. Adjust the parameters of the resonance-galvanometer scanner 10, observe the amplitude and phase changes of the signal output by the lock-in amplifier 20, and record the electrical signal output by the photomultiplier tube 19.

[0076] S10. Use the data acquisition card 21 to collect the electrical signal output by the photomultiplier tube 19, convert the voltage / current value of any point on the sample 12 to be measured into a digital signal, and upload it to the data analysis module.

[0077] S11. In the data analysis module, use Labview software and Matlab software to process the electrical signal output by the photomultiplier tube 19 to obtain the CARS microscopy imaging result of the sample 12 to be measured.

[0078] In the present invention, during the process of adjusting the parameters of the resonance-galvanometer scanner 10, observing the amplitude and phase changes of the signal output by the lock-in amplifier 20, and recording the electrical signal output by the photomultiplier tube 19, the specific steps are as follows:

[0079] When the relative distance between the objective lens 11 and the sample to be measured 12 remains unchanged, a coordinate system is established with the horizontal plane where the sample to be measured 12 is located as the coordinate plane xoy to determine the coordinates of the position of the light spot on the sample to be measured 12; when the value of the position coordinate of the light spot on the sample to be measured 12 y is a constant value, increase the voltage value applied to the two-dimensional precision translation stage x in the axis direction where the sample to be measured 12 is located. The x value of the light spot translates in steps of 1 μm, and simultaneously record the signals of the photomultiplier tube at 160 different x values.

[0080] When the value of the position coordinate of the light spot on the sample to be measured 12 y is y = y +1 μm, record the signals of the photomultiplier tube 19 at 160 different x values again simultaneously.

[0081] When the value of the position coordinate of the light spot on the sample to be measured 12 y is up to y = y +160 μm, record the signals of the photomultiplier tube 19 at 160 different x values again simultaneously. Scan a 160 μm × 160 μm area to obtain a 160 × 160 two-dimensional matrix, and record all the values at the grid points of the 160 × 160 two-dimensional matrix.

[0082] The CARS spectroscopy measurement and microscopic imaging device of the present invention utilizes a curved mirror to expand the femtosecond pulsed laser in space, and uses a tip to achieve laser spectral notch marking, thereby realizing a scheme for improving the existing single-beam CARS microscopic imaging with a tip. While reducing costs, the system becomes more compact and portable; secondly, by introducing a resonance-galvanometer scanner, the imaging speed of the device is effectively improved; secondly, by optimizing the positions of the various components for CARS spectroscopy detection, the purpose of gradually and orderly increasing the intensity of the generated CARS signal can be achieved; in addition, when using the near-infrared band as the incident laser pulse and preferably setting the laser pulse repetition frequency to 80 MHz and the pulse width to 10 fs, low-average-power CARS excitation can be achieved using the high peak power of the ultra-steep pulse, and the phototoxicity to biological samples is small; in summary, the device of the present invention has a simple structure, is inexpensive, has low energy consumption, and has the advantages of being compact, portable, and having a high imaging speed.

[0083] Specific experimental description:

[0084] Taking the test process of CARS spectroscopy measurement of tetrabromoethane and CARS microscopy imaging of rat tail tendon as an example, the CARS spectroscopy measurement and microscopy imaging device of the present invention will be described in detail;

[0085] The specific process is as follows:

[0086] A femtosecond pulsed 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 a dichroic mirror 2, and then passes through a pair of gratings 3, a curved mirror 4, a tip 5, and a first mirror 6 placed on a one-dimensional precision translation stage for notch shaping and dispersion compensation. After passing through a second mirror 7 and a third mirror 8, it then passes through a second beam splitter 22 and is divided into two beams. Among them, the reflected light of the second beam splitter 22 passes through a second spectrometer 23 to detect the notch position generated by the tip 5. The transmitted light of the second beam splitter 22 is filtered by an ultra-steep long-pass filter 9, and the filtered transmitted light is divided into two beams by a third beam splitter 24. Among them, the reflected light of the third beam splitter 24 enters a third spectrometer 26 for the ultra-steep edge position generated by the ultra-steep long-pass filter. The transmitted light of the third beam splitter 24 passes through a resonant galvanometer scanner 10, and then passes through an objective lens 11 placed on a three-dimensional precision translation stage and is focused on a sample to be measured 12. Among them, the sample to be measured 12 is placed in a sample cell on a two-dimensional precision translation stage. The heat dissipation signal formed after passing through the sample to be measured 12 is collected by a condenser lens 13 placed on another three-dimensional precision translation stage. Among them, the transmitted part passes through an ultra-steep short-pass filter 14 to filter out the incident laser. Then the light beam is divided into two beams by a first beam splitter 15. Among them, the transmitted light of the first beam splitter 15 passes through a lens 16 and is introduced into an optical fiber connected to a first spectrometer 17 for CARS spectroscopy measurement. The reflected light of the first beam splitter 15 is selectively reflected by a notch filter 18 and introduced into a photomultiplier tube 19 for CARS microscopy imaging. Finally, the electrical signals converted by the first spectrometer 17 and the photomultiplier tube 19 are extracted by a data acquisition card 21 and uploaded to a data analysis module for data processing.

[0087] Model description of the above components:

[0088] The model of the femtosecond pulsed laser 1 is: Thorlabs, OCTAVIUS-85M-HP; the model of the dichroic mirror 2 is: Thorlabs, DMLP650; the model of the grating 3 is: Milton Roy Company; the model of the curved mirror 4 is: Thorlabs, CM254-150-G01; the model of the tip 5 is: Nanopass33; the model of the first reflector 6 is: Thorlabs, ME2-M01; the models of the second reflector 7 and the third reflector 8 are both Thorlabs, UM10-AG-10; the model of the ultra-steep long-pass filter 9 is: Semrock LP02-785RE; the model of the objective lens 11 is: Newport, 20x, 0.4 NA; the model of the condenser lens 13 is: Edmund Optics, 0.5NA; the model of the ultra-steep short-pass filter 14 is: Semrock SP01-785RU; the models of the first beam splitter 15, the second beam splitter 22 and the third beam splitter 24 are all: Thorlabs, ME2-M01; the model of the lens 16 is: Thorlabs, AC254-030-AB; the model of the first spectrometer 17 is: Jobin Yvon Triax 320; the model of the notch filter 18 is: Unaxis Blazers; the models of the second spectrometer 23 and the third spectrometer 25 are both 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.

[0089] It should be noted that when using tetrabromoethane as the sample 12 to perform CARS spectroscopy measurement, start the laser and all spectrometers, and observe the incident laser spectrum, the position of the tip, and the position of the ultra-steep edge, as shown in the appendix Figure 2 ; among them, appendix Figure 2 (a) is the incident laser spectrum measured by the second spectrometer when the tip is at 779.9 nm; appendix Figure 2 (b) is the incident laser spectrum measured by the third spectrometer when the tip is at 775 nm; appendix Figure 2 (c) is the incident laser spectrum measured by the third spectrometer when the tip is at 779.9 nm; by measuring the original CARS spectrum when the tip is at 779.6 nm, then recording the original CARS spectrum when the tip is at 779.9 nm, and then based on the original CARS spectrum when the tip is at 779.6 nm and the original CARS spectrum when the tip is at 779.9 nm, obtain the Raman spectrum of tetrabromoethane, that is, obtain the CARS spectroscopy measurement of tetrabromoethane, as shown in the appendix Figure 3 ; among them, appendix Figure 3(a) The original CARS spectrum of tetrabromoethane measured by the third spectrometer when the tip was at 779.6 nm and 779.9 nm; attached Figure 3 (b) The Raman spectrum of tetrabromoethane obtained by processing the original CARS spectrum.

[0090] It should be noted that when the rat tail tendon was used as the sample 12 to be measured for CARS microscopy, by selecting the reflection wavelength of the notch filter 18, it was possible to meet the requirements for CARS microscopy of the sample at different vibration frequencies; when the reflection wavelength of the notch filter 18 was 784.6 nm, microscopy of the rat tail tendon was performed, as shown in the attachment Figure 4 shown.

[0091] For the CARS spectrum measurement and microscopy device and method of the present invention, by placing a steel tip on the dispersion compensator and using the method of tip occlusion to generate a notch in the laser spectrum to achieve probe light marking, replacing the notch filter realized by precision coating of quartz glass, the experimental cost is reduced at the same time; a resonant-galvanometer scanner (12 kHz) is used instead of the ordinary galvanometer scanner (1 kHz) in the traditional single-beam CARS scheme, and the single-pixel imaging speed is increased to less than 100 microseconds. Compared with the traditional single-beam CARS microscopy imaging speed based on notch filtering, it can be increased by 10 times and can be applied to fast imaging in the field of biomedicine.

[0092] In the present invention, the incident laser is modulated at a high frequency above 10 kHz by using a resonant-galvanometer scanner, so as to modulate the resonant term in the CARS signal at a high frequency; since the resonant CARS in the generated CARS signal is only related to the tip position, and the non-resonant CARS is not related to the tip position and can be used as a local oscillator; the non-resonant signal is removed by lock-in amplification, and the weaker resonant signal in the CARS signal is extracted; by slightly adjusting the position of the tip, two groups of original CARS spectra are measured, and after differentiating and normalizing the two, the low-frequency vibration spectrum or Raman spectrum, including the vibration spectrum in the terahertz band, can be obtained; secondly, by introducing an ultra-steep long-pass filter and an ultra-steep short-pass filter, it is ensured that the incident laser is infinitely close to the detected CARS signal, so that the detection of the vibration spectrum in the terahertz band can be realized.

[0093] It should be noted that when the diameter of the tip is set to 0.12 mm, a high spectral resolution can be ensured; since biological macromolecules, such as proteins and DNA, have large-amplitude vibration modes in the terahertz band; therefore, a CARS system with high spectral resolution and fast microscopic imaging can be realized by using the tip and the resonant galvanometer scanner; the single-beam CARS spectral resolution based on the tip depends on the thickness of the tip, the thinner the tip, the higher the spectral resolution; that is to say, the single-beam CARS spectral resolution depends on the width of the notch generated on the laser spectrum, the narrower the notch, the higher the spectral resolution; in the prior art, the single-beam CARS spectral resolution based on the notch filter is 14 wave numbers, while in the present invention, when the diameter of the tip is 0.3 mm, its spectral resolution can reach below 10 wave numbers, achieving the purpose of optimizing the single-beam CARS spectral resolution; secondly, since the length of the tip is controlled within 1-20 mm and the mass is about 0.4 g, it is smaller and lighter than the notch filter; at the same time, due to the tip being small enough, the power loss of the laser when generating the notch is less than half of that of the notch filter, so the energy consumption is low; in addition, the tip only needs simple grinding and does not require complex processes such as precision coating, so the cost is low; secondly, to generate a notch on the laser spectrum with the tip, the tip only needs to be fixed on a one-dimensional translation stage and does not require precision rotation, eliminating the precision rotary stage, so the operation is relatively simple.

[0094] The above embodiments are only 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 only by this embodiment, but also includes any changes, substitutions and other implementation manners that are easily conceivable by those skilled in the art within the technical scope disclosed by the present invention.

Claims

1. A CARS spectral 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 passed through a dichroic mirror (2), a grating (3), a curved mirror (4), a needle tip (5), a first reflector (6), a second reflector (7), a third reflector (8), an ultra-steep long-pass filter (9), a resonance-galvanometer scanner (10) and an objective lens (11), and then irradiated onto a sample to be tested (12) placed on a two-dimensional precision translation stage, thereby forming a scattering signal; The scattered signal passes through the transmission part of the sample to be measured (12) and passes through a condenser (13) and an ultra-steep short-pass filter (14) in sequence, and then is split into two paths through a first beam splitter (15); one path passes through a lens (16) and enters a first spectrometer (17) for CARS spectrum measurement; the other path passes through a notch filter (18) and a photomultiplier tube (19) and enters a lock-in amplifier (20) for CARS microscopic imaging; The dichroic mirror (2), the grating (3), the curved mirror (4) and the needle tip (5) are sequentially arranged on the optical path of the laser pulse, and the dispersion compensation of the laser pulse is achieved by adjusting the relative position of the grating (3) and the curved mirror (4), thereby forming a compensated laser pulse; The needle tip (5) is arranged on a one-dimensional translation stage and is used for notch shaping of the laser spectrum, that is, notch marking is performed on the compensated laser pulse to form a notch-marked laser pulse; the needle tip (5) adopts a steel needle tip structure; The first reflector (6) is arranged on the optical path of the laser pulse after the notch mark, and is used to reflect the laser pulse after the notch mark to form a first reflected light beam; the first reflected light beam passes through the curved mirror (4), the grating (3) and the dichroic mirror (2) in sequence and then enters the second reflector (7).

2. A CARS spectral measurement and microscopic imaging device according to claim 1, characterized in that: The needle tip (5) has a diameter of 0.1-0.5 mm and a length of 1-20 mm.

3. A CARS spectral measurement and microscopic imaging device according to claim 1, characterized in that: It also includes a second beam splitter (22) and a second spectrometer (23); The second beam splitter (22) is arranged between the third reflector (8) and the ultra-steep long-pass filter (9), and the second spectrometer (23) is arranged on the side of the second beam splitter (22); The reflected light path of the third reflector (8) forms a reflected light path and a transmitted light path after passing through the second beam splitter (22); the reflected light path formed by the second beam splitter (22) enters the second spectrometer (23); and the transmitted light path formed by the second beam splitter (22) enters the ultra-steep long-pass filter (9).

4. A CARS spectral measurement and microscopic imaging device according to claim 1, characterized in that: It also includes a third beam splitter (24) and a third spectrometer (25); The third beam splitter (24) is arranged between the ultra-steep long-pass filter (9) and the resonance-galvanometer scanner (10), and the third spectrometer (25) is arranged on the side of the third beam splitter (24); The transmitted light of the ultra-steep long-pass filter (9) forms a reflected light path and a transmitted light path after passing through a third beam splitter (24); the reflected light path formed by the third beam splitter (24) enters the third spectrometer (25); and the transmitted light path formed by the third beam splitter (24) enters the resonance-galvanometer scanner (10).

5. A CARS spectral measurement and microscopic imaging device according to claim 1, characterized in that: The blazing wavelength of the grating (3) is 750 nm, wherein 1200 notches are arranged per millimeter.

6. A CARS spectral measurement and microscopic imaging method, characterized in that: Using the CARS spectroscopy measurement and microscopic imaging device as described in any one of claims 1 to 5, the CARS spectroscopy measurement and microscopic imaging method comprises a CARS spectroscopy measurement step; The CARS spectrum measurement steps are as follows: Coarsely adjusting the focus of the objective lens (11) so that the laser is focused on the sample to be measured (12) until the original CARS signal is observed in the first spectrometer (17); adjusting the positions of the first reflector (6), the second reflector (7), the third reflector (8) and the condenser (13) so that the intensity of the CARS spectrum measured in the first spectrometer (17) is maximized; Adjusting the relative position of the curved mirror (4) and the grating (3) so that the dispersion of the laser light on the sample (12) to be measured is compensated until the original CARS signal no longer increases; Observe the CARS spectrum intensity measured in the first spectrometer (17), and adjust the position of the first beam splitter (15) until the original CARS signal no longer increases; Adjusting the optical fiber angle of the first spectrometer (17) until the original CARS signal no longer increases; The needle tip (5) is adjusted to different positions to generate two notches of different wavelength sizes, and the original CARS spectrum data under the notches of the two different wavelength sizes are respectively recorded using the first spectrometer (17); and the CARS spectrum measurement result of the sample to be tested (12) is obtained based on the original CARS spectrum data under the notches of the two different wavelength sizes.

7. A CARS spectral measurement and microscopic imaging method according to claim 6, characterized in that: The grating (3) is mounted on a one-dimensional precision translation stage; The process of adjusting the relative position of the curved mirror (4) and the grating (3) is specifically as follows: The one-dimensional precision translation stage is adjusted to change the position of the grating (3), thereby adjusting the relative position of the curved mirror (4) and the grating (3).

8. A CARS spectral measurement and microscopic imaging method according to claim 6, characterized in that: The process of adjusting the needle tip (5) to different positions to generate two gaps of different wavelength sizes, and using the first spectrometer (17) to respectively record the original CARS spectrum data under the gaps of two different wavelength sizes is as follows: Adjusting the angle of the ultra-steep long-pass filter (9) to adjust the cutoff edge wavelength of the ultra-steep long-pass filter to a preset edge wavelength; The position of the needle tip (5) is adjusted using a one-dimensional translation stage until the wavelength of a notch formed in the needle tip (5) is a preset first notch wavelength; and original CARS spectrum data is recorded using a first spectrometer (17) to obtain original CARS spectrum data under the first wavelength notch. ; The position of the needle tip (5) is adjusted again using the one-dimensional translation stage until the wavelength of the notch formed by the needle tip (5) is a preset second notch wavelength; the original CARS spectrum data is recorded using the first spectrometer (17) to obtain the original CARS spectrum data under the second wavelength notch. .

9. A CARS spectral measurement and microscopic imaging method according to claim 8, characterized in that: The preset edge wavelength is 779 nm, the preset first notch wavelength is 779.6 nm, and the preset second notch wavelength is 779.9 nm.

10. A CARS spectrum measurement and microscopic imaging method according to claim 6, characterized in that: The CARS 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 (18) to determine the characteristic vibration mode of the sample (12) to be tested; adjusting the position of the photomultiplier tube (19) so that the voltage value or current value of the electrical signal output by the photomultiplier tube (19) is maximized; Adjusting the parameters of the resonance-galvanometer scanner (10), observing the amplitude and phase change of the signal output by the lock-in amplifier (20), and recording the electrical signal output by the photomultiplier tube (19); The electrical signal output by the photomultiplier tube (19) is processed to obtain a CARS microscopic imaging result of the sample to be tested (12).

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