CN107167455A - Light splitting pupil laser differential confocal CARS micro-spectrometer method and devices - Google Patents

Abstract

The invention belongs to microspectrum imaging detection technical field, it is related to a kind of light splitting pupil laser differential confocal CARS micro-spectrometer method and devices.The core concept of the present invention is twin-laser as light source activation Reyleith scanttering light and the CARS light for being loaded with sample spectral characteristic, lossless separation is carried out to Reyleith scanttering light and CARS light using dichroic optical system, wherein Reyleith scanttering light carries out geometry detection and positioning, and CARS light carries out spectrographic detection.The present invention utilizes accurate this corresponding characteristic in light splitting pupil laser differential confocal curve zero crossing and focal position, accurate capture and localized excitation light spot focus position, the spectrographic detection of high-precision geometry detection and high-space resolution is realized, a kind of method and apparatus of achievable sample microcell high-space resolution spectrographic detection are constituted.By combining CARS microtechnics, the time of what is inspired the be loaded with Raman diffused light of sample message is short than traditional spontaneous Raman effect, and energy quick nondestructive is detected to sample.The present invention has the advantages that accurate positioning, high-space resolution, lossless detection, spectral detectivity are high, is that microscopic spectrum detection and dimensional measurement provide a kind of new approach.

Description

Split pupil laser differential confocal CARS microspectral testing method and device

technical field

The invention belongs to the technical field of microspectral imaging, and relates to a split-pupil laser differential confocal CARS microspectral testing method and device, which can be used to quickly detect micro-area anti-Stokes scattering (CARS) spectra of various samples, High spatial resolution imaging and detection can be realized.

technical background

Optical microscopes are widely used in the fields of biomedicine and material science. With the rapid development of modern science, the requirements for microscopic imaging have shifted from structural imaging to functional imaging. In 1990, the successful application of confocal Raman spectroscopy greatly improved the possibility of exploring the specific composition and morphology of tiny objects. It combines confocal microscopy technology and Raman spectroscopy technology. It has the high-resolution tomographic imaging characteristics of confocal microscopy, and has the ability of non-destructive detection and spectral analysis. It has become an important technical means for material structure measurement and analysis. , Widely used in physics, chemistry, biomedicine, material science, petrochemical, food, medicine, criminal investigation and other fields.

The traditional spontaneous Raman scattering imaging technology has extremely weak emission signals due to the characteristics of Raman scattering itself. Even with high-intensity laser excitation, it still takes a long time to obtain a spectral image with good contrast. This long-term effect limits the application of Raman microscopy in the biological field. The coherent anti-Stokes Raman scattering (CARS) process based on the coherent Raman effect can greatly enhance the Raman signal, thus realizing fast detection. The coherent Raman effect is to lock the molecules on the vibrational energy level through the excited light. The intensity of the vibration signal generated by this method has a nonlinear relationship with the intensity of the excitation light, which can generate a strong signal, also known as coherence. Nonlinear Raman spectroscopy. It has strong energy conversion efficiency, short exposure time, and relatively little damage to the sample. At the same time, its scattering has a certain directionality, and it is easy to separate from stray light.

The generation of coherent anti-Stokes Raman scattering (CARS) is a third-order nonlinear optical process, which requires pump light, Stokes light and probe light. Generally speaking, in order to reduce the number of light sources and simplify the process, the pump light is often used instead of the probe light. The relationship between them is shown in Figure 2. When the pump light (w _p ) and the Stokes light (w _s ) When the frequency difference of is matched with the vibration frequency of the Raman active molecule, the CARS light w _as will be excited, where w _as =2w _p -w _s . The generation process of CARS light includes the interaction process between the vibration mode of specific Raman active molecules and the incident light field that leads to the vibrational transition of molecules from the ground state to the excited state. Its energy level diagram is shown in Figure 3. Figure 3(a) shows the contribution of Raman resonance and non-resonance single-photon enhancement to the CARS process, and Figure 3(b) shows the contribution of Raman resonance and non-resonance two-photon enhancement to the CARS process; when w _p and w _s When the frequency difference matches the vibration frequency of the Raman active molecule, the excited signal is resonantly enhanced;

Traditional CARS microscopy mostly uses two single-wavelength lasers, which can only obtain spectral information of a specific spectrum, and traditional CARS microscopy does not emphasize the system's fixed-focus capability, resulting in the actual spectral detection position often being in an out-of-focus position. Even the out-of-focus position can excite the Raman spectrum of the sample and be detected by the spectrometer behind the pinhole, but the intensity cannot reasonably represent the correct spectral signal intensity at this point. In the CARS microscope system, the best spatial resolution and the best spectral detection ability can only be obtained when the system is precisely focused.

The above reasons limit the ability of the CARS microscopic system to detect micro-region spectra, and restrict its application in finer micro-region spectral testing and analysis occasions. Based on the above situation, the present invention proposes to detect the Rayleigh light collected by the system that is 10 ³ to 10 ⁶ times stronger than the Raman scattered light of the sample surface, so that it can be organically integrated with the spectral detection system to perform spatial position information Synchronous detection of spectral information and spectral information to achieve high spatial resolution and high spectral resolution of split-pupil laser differential confocal CARS spectrum imaging and detection.

The core idea of the patent of the present invention is to select a supercontinuum pulsed laser and a single-wavelength pulsed laser as the excitation light source to expand the range of the excitation spectrum and increase the excitation intensity of the spectrum; The offset can cause the axial response characteristic curve of the split-pupil confocal microscope system to produce a phase shift characteristic, and the focal spot is symmetrically divided, and the two-way signals are differentially processed to realize differential detection. According to the differential principle, it realizes bipolar absolute zero point tracking measurement, precise focusing, and then realizes high spatial resolution; after precise focusing, spectral detection is performed to obtain the best spectral resolution capability.

Contents of the invention

The purpose of the present invention is to overcome the deficiencies of the prior art, and propose a high spatial resolution split-pupil laser differential confocal CARS microspectral testing method and its device.

The present invention is achieved through the following technical solutions. The split-pupil laser differential confocal CARS microspectral testing method comprises the following steps:

a) The supercontinuum laser is emitted by the supercontinuum laser. After passing through the bandpass filter, it merges with the single-wavelength laser emitted by the single-wavelength laser through the first dichroic mirror. By adjusting the optical path, the timing of the two beams is consistent and the space coincides. (Single-wavelength laser is enveloped in continuum laser); the mixed beam converges on the sample to be measured through the illumination pupil and the microscope objective lens, and excites Rayleigh light and CARS light carrying the spectral characteristics of the sample to be measured;

b) CARS light and Rayleigh light are divided into two beams after passing through the second dichroic mirror, wherein the beam containing CARS light enters the spectral detection unit, and the other beam containing Rayleigh light enters the split-pupil laser differential confocal detection unit; In the spectral detection unit, the light beam containing CARS light first passes through a bandpass filter to filter out the non-CARS interfering light in the light beam, and then converges through the first pinhole through the first converging mirror, filters the ambient light, and then is converged by the second The mirror converges into the spectrometer to obtain the CARS spectrum information; another beam carrying Rayleigh light passes through the third converging mirror and is detected by the light intensity acquisition system for focal spot segmentation to obtain the signals corresponding to the detection area A and detection area B respectively.

c) After performing differential subtraction processing on the signals of the two detection areas A and B, the differential confocal curve is obtained, and the "zero crossing point" of the split-pupil laser differential confocal response curve is used to accurately correspond to the focal position of the measurement objective lens , to accurately capture the focus position of the excitation spot through "zero-crossing" triggering, and realize geometric detection and spatial positioning with high spatial resolution.

d) The position of the surface of the sample to be measured (focus of the excitation spot) is obtained through computer processing, and the laser is focused on the surface of the sample to be measured by controlling the movement of the high-precision three-dimensional scanning translation stage, and the CARS spectral information of this point is obtained through the spectral detection unit.

e) Separate processing of Rayleigh light signals can realize high spatial resolution three-dimensional scale tomography; separate processing of CARS spectral signals can obtain spectral images; simultaneous processing of Rayleigh signals and CARS spectral signals can achieve high spatial resolution of the map layer Analytical imaging, that is, to realize the integrated imaging and detection of the geometric position information and spectral information of the measured sample.

In particular, in the method of the present invention, the illumination pupil and the collection pupil may be circular, D-shaped or other shapes.

In particular, in the method of the present invention, the excitation light beam includes polarized light beams such as linearly polarized light, circularly polarized light, and radially polarized light, and structured light beams generated by techniques such as pupil filtering, thereby improving the system spectral signal-to-noise ratio and the lateral resolution of the system. Rate.

Particularly, in the method of the present invention, by matching the filters of different spectral bands, select the Stokes light of different spectral bands, can realize the spectral detection of different spectral bands; Wherein, band-pass filter and band-pass filter The filter band of the light sheet is symmetrical about the central wavelength of the single-wavelength laser.

In particular, in the method of the present invention, the laser emission unit can also be realized by using a single-wavelength laser plus a photonic crystal fiber for spectrum broadening. In addition, the spectrometer in the spectrum detection unit is replaced by a photoelectric point detector, and the rotating polarizer can realize spectrum scanning. Output, and then excite the CARS spectrum and get the CARS spectrum signal detected by the photoelectric point detector;

The present invention provides a split-pupil laser differential confocal CARS micro-spectroscopy test device, comprising: a spectrum excitation unit, a laser emission unit located in the incident direction of the spectrum excitation unit, a dichroic unit located in the exit direction of the spectrum excitation unit, and a The spectral detection unit in the transmission direction of the chromatic unit, the differential confocal detection unit in the reflection direction of the dichroic unit, and the computer controlling the entire system. The laser emission unit consists of a single-wavelength pulse laser, a supercontinuum pulse laser, a bandpass filter and a first dichroic mirror; the spectrum excitation unit consists of an illumination pupil, a collection pupil, a measurement objective lens, a sample to be tested and a high-precision The three-dimensional scanning translation stage is composed of; the dichroic unit is the second dichroic mirror; the spectral detection unit is composed of a bandpass filter, the first converging mirror, the first pinhole, the second converging mirror and a spectrometer; the laser differential common The focal detection unit is composed of a third converging mirror and a light intensity collection system.

In the device of the present invention, the light intensity collection system can adopt the method of combining double pinholes and two-quadrant detectors to realize segmented detection of the Airy disk.

In the device of the present invention, the light intensity collection system can use a CCD detector, and the detection of the Airy disc can be segmented and detected by setting the position and size of the detection area on the CCD detection surface.

In the device of the present invention, the light intensity collection system can use conductive optical fibers, and by placing two optical fibers symmetrically about the optical axis on the focal plane of the third converging mirror, the split detection of the Airy disk can be realized.

In the device of the present invention, the Airy disk detected by the light intensity collection system can be enlarged by adding a relay magnification lens, so as to improve the collection accuracy of the split-pupil laser differential confocal measurement device.

Beneficial effect

The inventive method has the following innovations compared with the prior art:

1. The present invention organically combines the split-pupil laser differential confocal microscopy technology with the CARS spectral detection technology, and utilizes the characteristic that the "zero point" position of the split-pupil laser differential confocal curve precisely corresponds to the focal position of the microscope objective lens to accurately capture the focus of the excitation spot position and detect spectral information, so as to realize spectral tomography and detection with high spatial resolution.

2. In the present invention, a dichroic mirror is used to separate Rayleigh light from CARS light carrying sample information, wherein Rayleigh light enters the split pupil laser differential confocal detection unit to realize geometric position detection, and CARS light enters the spectral detection unit to realize CARS Spectral detection, through precise focusing to capture the spectral information of the focal point of the excitation spot, improves the sensitivity of the system's spectral detection. In addition, the angle of the dichroic light-splitting device can be adjusted according to needs, which is convenient for structural installation and adjustment.

3. The present invention combines the split-pupil laser differential confocal microscope system and the CARS spectral imaging system in structure and function, which can realize the tomographic imaging of the geometric parameters of the micro-area of the sample, and can also realize the spectral detection of the micro-area of the sample. Implement spectral tomography.

The inventive method has the following significant advantages compared with the prior art:

1. The split-pupil laser differential confocal detection method adopted in the present invention utilizes the characteristics of differential subtraction, noise cancellation, and strong anti-environment interference ability.

2. The present invention uses split-pupil laser differential confocal technology to perform high-precision positioning of the measurement focus spot, and performs real-time tracking and measurement of the focus position, eliminating environmental influences such as temperature and disturbance, and realizing that CARS spectrum detection always accurately corresponds to the minimum excitation focus spot The sample spectrum of the area can greatly improve the micro-area spectral detection ability and geometric position detection ability of the existing CARS microscope.

3. The present invention uses a supercontinuum laser matched with a single-wavelength laser as an excitation light source, which can realize broadband CARS spectrum detection.

Description of drawings

Fig. 1 is abstract accompanying drawing, i.e. basic implementation figure of the present invention;

Figure 2 is a schematic diagram of coherent anti-Stokes (CARS) optical excitation;

Figure 3 is a diagram of the relationship between CARS light, pump light, and Stokes light

Fig. 4 is a traditional polarization detection microscopic light path diagram;

Figure 5 is a graph of the response curve of the split-pupil laser differential confocal;

6 is a schematic diagram of a D-shaped pupil-divided laser differential confocal CARS microscopic testing method;

7 is a schematic diagram of a microscopic test method for split-pupil laser differential confocal CARS combined with a pupil filter;

Fig. 8 is a schematic diagram of a split-pupil laser differential confocal CARS microscopic test method using a double pinhole and a two-quadrant detector;

9 is a schematic diagram of a microscopic testing method for split-pupil laser differential confocal CARS using a CCD detector;

Fig. 10 is a schematic diagram of a split-pupil laser differential confocal CARS microscopic testing method using optical fiber for detection;

Fig. 11 is a schematic diagram of a split-pupil laser differential confocal CARS microscopic testing device with a detection focal spot amplification system;

Fig. 12 is a schematic diagram of a split-pupil laser differential confocal CARS microscopic test method with a single laser light source;

Fig. 13 is a schematic diagram of the high spatial resolution split-pupil laser differential confocal CARS microscopic testing method and device, that is, the diagram for the embodiment.

In the figure, 1-laser emitting unit, 2-single-wavelength laser source, 3-supercontinuum laser source, 4-bandpass filter, 5-first dichroic mirror, 6-measurement objective lens, 7-illumination light Pupil, 8-Collection pupil, 9-Sample under test, 10-High-precision three-dimensional scanning translation stage, 11-Second dichroic mirror, 12-Band pass filter, 13-First condenser lens, 14-First Pinhole, 15-second condenser, 16-spectrometer, 17-spectral detection unit, 18-divided pupil laser differential confocal detection unit, 19-third condenser, 20-light intensity acquisition system, 21-detection area A Curve, 22-curve of detection area B, 23-first off-axis confocal axial curve, 24-second off-axis confocal axial curve, 25-differential curve, 26-pupil filter, 27-double Pinhole, 28-two-quadrant detector, 29-CCD detector, 30-guide fiber 1, 31-guide fiber 2, 32-relay magnifying lens, 33-polarization beam splitter prism, 34-polarizer, 35-photonic crystal Optical fiber, 36-first mirror, 37-optical delay line, 38-second mirror, 39-photoelectric point detector, 40-computer;

detailed description

The present invention will be described in further detail below in conjunction with the accompanying drawings and embodiments.

Fig. 1 is a schematic diagram of the microspectral detection method of the split-pupil laser differential confocal CARS. First, a single-wavelength pulsed laser (2) is selected as the pump light source and probe light source, which emits pump light (probe light), and then a supercontinuum pulse laser (3) with the same frequency is selected as the Stokes The Sri Lankan light source, after passing through the band-pass filter (4), obtains the continuum laser in the required wavelength range, and by adjusting the optical structure, the time of the two laser beams passing through the first dichroic mirror (5) is consistent, and the space overlaps; The final light is focused on the measured sample (9) through the illumination pupil (7) and the measuring objective lens (6); because in the case of tight focusing, the phase matching condition is easy to meet, the Rayleigh light and the measured The CARS light of the spectral characteristics of the sample; CARS light and Rayleigh light respectively arrive at the spectral detection unit (17) and the split-pupil laser differential confocal detection unit (18) after passing through the second dichroic mirror (11); wherein, the spectral detection unit (17) performing spectrum detection on the CARS light, and the split-pupil laser differential confocal detection unit (18) performing geometric position detection on the Rayleigh light.

In the split-pupil laser differential confocal detection unit (18), the Rayleigh light is converged by the third converging mirror, and is divided and detected by the light intensity acquisition system to obtain the first micro-area of the Airy disk and the second micro-area of the Airy disk Intensity characteristic curve, that is, the first off-axis confocal axial curve (23) and the second off-axis confocal axial curve (24); the first off-axis confocal axial curve (23) and the second off-axis confocal The focal axis curve (24) is subtracted to obtain the split-pupil laser differential confocal curve (25), as shown in FIG. 5 .

Replacing the circular illumination pupil (7) and collection pupil (8) with other shapes, such as D-shape, constitutes a D-shape split-pupil laser differential confocal CARS test method, as shown in Figure 6.

The spatial resolution of spectral detection is improved by adding a pupil filter (26), that is, a split-pupil laser differential confocal CARS test method with pupil filter added is formed, as shown in FIG. 7 .

The light intensity acquisition system (20) can adopt the method of combining double pinholes (27) and two-quadrant detectors (28) to realize segmented detection of the Airy disk, as shown in FIG. 8 .

The light intensity acquisition system (20) can use a CCD detector (29), and by changing the parameters of the tiny area set on the detection focal plane to match the reflectivity of different samples, the segmented detection of the Airy disk can be realized, thereby expanding Its application fields are shown in Figure 9.

The light intensity acquisition system can use conductive optical fibers, and by placing two optical fibers (30, 31) symmetrically along the optical axis at the focal plane of the third converging mirror (19), the segmentation detection of Airy disks can be realized, as shown in Figure 10 Show.

An amplification system (32) can be added to the split-pupil laser differential confocal detection system to improve the acquisition accuracy of the split-pupil laser differential confocal measurement device, as shown in FIG. 11 .

Figure 12 is a schematic diagram of a single-laser laser differential confocal CARS microscopic test method, the function is to change the dual-laser input of the laser emitting unit into a single-laser input to reduce costs; a single-wavelength pulsed laser emits a single-wavelength laser, which passes through a polarization beam splitter ( 33) Light splitting, the transmission part enters the photonic crystal fiber (35) through the polarizer (34) to carry out spectral band broadening and uses a band-pass filter to intercept specific required wavelengths, and the reflection part passes through the first reflector (36), optical delay line ( 37) and the second reflector (38) are coupled with the broadened continuum laser at the first dichroic mirror (5), outputting a mixed light beam with consistent space and time, and performing CARS spectrum excitation on the measured sample . Wherein, the function of the optical delay line (37) is to ensure that the timing of the two laser beams coincides. Further replace the spectrometer (16) in the spectrum detection unit (17) with a photoelectric point detector (39), rotate the polarizer (34) to change the beam polarization state and make the spectral line of the photonic crystal fiber (37) output wavelength continuously change as the Si Tox light, and then realize the wide-band CARS spectrum measurement.

Example

In this embodiment, a picosecond laser with a wavelength of 532nm is used as the pump light source and a probe light source, and a supercontinuum picosecond laser with the same repetition rate is used as the Stokes light source with a bandpass filter of 550-650nm , mixed and emitted under the conditions of space coincidence and time consistency, after the structured beam is obtained through the pupil filter, the illumination pupil and the measurement objective lens are tightly focused on the sample, and the phase matching condition is satisfied at this time, and the excitation wavelength range is 450 Anti-Stokes light (CARS) at ~515 nm and Rayleigh light at 532 nm.

As shown in Figure 13, it is a split-pupil laser differential confocal CARS micro-spectroscopy test device, and its test steps are as follows:

First, in the laser emitting unit (1), the continuum laser emitted by the supercontinuum laser (3) is filtered by a bandpass filter (4) to obtain a broadband laser of 550-650, and then combined with a single wavelength (532nm) The monochromatic laser light emitted by the laser (2) converges at the first dichroic mirror (5) to form a mixed beam, wherein the repetition frequency of the two laser beams is the same, and the arrival time of the first dichroic mirror (5) is consistent, and the beams converge After that, it can be completely overlapped (the pump spot is completely enveloped by the Stokes spot); the mixed beam passes through the pupil filter to generate a structured beam; the structured beam is tightly focused on the measured sample (9) through the illumination pupil and the measurement objective lens , to excite Rayleigh light and CARS light carrying the spectral characteristics of the measured sample (9).

At this time, the scanning of the sample can be completed in the following ways: realize scanning in the x-y-z direction by a high-precision three-dimensional scanning translation stage (10), or add a galvanometer scanning structure to the optical path after the laser is emitted to realize scanning in the x-y direction, and realize it through PZT axial scan.

The light beam reflected back by the sample to be tested includes Stokes light λ _s , pump light λ ₀ , Rayleigh light λ ₀ , and CARS light λ _as ; wherein, CARS light λ _as and Stokes light λ _s enter the spectrum detection unit (17), 532nm pump light and Rayleigh light are reflected by the second dichroic mirror (11) and enter the split-pupil laser differential confocal detection unit (18). In the spectral detection unit (17), the light mixed by the Stokes light λ _s and the CARS light λ _as first passes through the bandpass filter (12) of 450-515nm and only retains the CARS light, and then passes through the first converging The mirror (13) converges through the first pinhole (14), filters the ambient light and then converges into the spectrometer (16) by the second converging mirror (15), thereby detecting and obtaining the CARS spectrum I(λ), where λ is the sample to be tested (9) The wavelength of the CARS light excited by the excited light. In the sub-pupil laser differential confocal detection unit (18), the Rayleigh light λ ₀ is converged by the third converging mirror, and is received by the CCD detector (29), by setting the detection area symmetrically along the optical axis on the CCD detection surface The position and size of the Airy disc are segmented and detected to obtain the first off-axis confocal axial curve (23) and the second off-axis confocal axial curve (24). Perform difference processing on the two signals to obtain a differential signal (25). The "zero point" of the split-pupil laser differential confocal response curve (25) corresponds precisely to the focus of the excitation beam, and the height information of the surface of the sample (9) to be measured is obtained through the "zero point" of the response curve (25), combined with the connection height The position information fed back by the displacement sensor of the precision three-dimensional scanning translation stage (10) reconstructs the surface three-dimensional topography I(x, y, z) of the measured sample (9) through the computer (40).

The computer (40) controls the high-precision three-dimensional scanning translation stage (10), so that the laser light is precisely focused on the surface of the measured sample (9), and the CARS spectrum I(λ) that can correctly characterize the spectral characteristics of the measured sample is excited, and the spectral detection unit (17) Acquisition, the position information I(x, y, z) and the spectral information I(λ) are fused by the computer (40), and the three-dimensional reconstruction and spectral information fusion I(x, y) of the measured sample (9) are completed , z, r). In addition, the computer system runs through the entire system, and the computer (40) is used to realize the displacement control of the high-precision three-dimensional scanning translation stage, the acquisition and processing of the split-pupil laser differential confocal signal and the CARS spectral signal, and the data fusion processing.

Above, along the laser emission direction, place the laser emission unit (1), the illumination pupil (7), the microscope objective lens (6), the sample to be tested (9), the high-precision three-dimensional translation stage (10), and the collection pupil ( 8), the second dichroic mirror (11), the spectral detection unit (17) is placed in the transmission direction of the second dichroic mirror (11), and the split pupil laser differential common is placed in the reflection direction of the dichroic mirror (11). Focus detection unit (18). The laser emission unit (1) comprises a supercontinuum laser (3), a bandpass filter (4) and a first dichroic mirror (5) and a single-wavelength pulsed laser (2); Place bandpass filter (12), the first converging mirror (13), the first pinhole (14), the second converging mirror (15), spectrometer (16); in the split pupil laser differential confocal detection unit ( 18), place the third converging mirror (19) and CCD detector (29) respectively. In the whole system, the single-wavelength pulse laser (2), the supercontinuum laser (3), the high-precision three-dimensional scanning translation stage (10), the spectrometer (16), and the CCD detector (29) are all controlled by the computer (40), The three-dimensional position information and spectral information obtained by the system are also fused and processed by the computer (40).

The specific embodiment of the present invention has been described above in conjunction with the accompanying drawings, but these descriptions can not be interpreted as limiting the scope of the present invention, the protection scope of the present invention is defined by the appended claims, any claims on the basis of the present invention The changes made are within the protection scope of the present invention.

Claims (10)

1. A split-pupil laser differential confocal CARS microspectral testing method and device, is characterized in that comprising the following steps: a) The supercontinuum laser is emitted by the supercontinuum laser (3), passes through the bandpass filter (4) and merges with the single-wavelength laser emitted by the single-wavelength laser (2) through the first dichroic mirror (5), By adjusting the optical path, the timing of the two beams is consistent and the space coincides (single-wavelength laser is enveloped in the continuum laser); the mixed beam is converged on the sample (9) to be measured through the illumination pupil (7) and the microscope objective lens (6), Excite Rayleigh light and CARS light carrying the spectral characteristics of the sample to be measured (9); b) CARS light and Rayleigh light are divided into two beams after passing through the microscope objective lens, collecting pupil (8) and second dichroic mirror (11), wherein the light beam containing CARS light enters the spectral detection unit (17), and the other beam The light beam that contains Rayleigh light enters the sub-pupil laser differential confocal detection unit (18); in the spectral detection unit (17), the light beam that contains CARS light first passes through a band-pass filter (12) to filter out the non- The CARS interfering light is then converged by the first converging mirror (13), and the ambient light is blocked by the first pinhole (14), so that the ambient light interference is reduced and then converged into the spectrometer (16) by the second converging mirror (15) to obtain CARS spectrum information; another beam carrying Rayleigh light is converged by the third converging mirror (19), received by the light intensity acquisition system (20), and the focal spot is segmented to obtain the detection area A (21) and the detection area The signal corresponding to B(22). c) The differential confocal curve (25) is obtained after normalizing and subtracting the signal of the measurement area A (21) and the signal of the detection area B (22), using the "zero crossing point" of the split-pupil laser differential confocal response curve It precisely corresponds to the focal position of the measurement objective lens, and the focal position of the excitation spot is accurately captured through the "zero-crossing point" trigger to achieve geometric detection and spatial positioning with high spatial resolution. d) Obtain the position of the surface of the measured sample (focus of the excitation spot) through computer (40), control the high-precision three-dimensional scanning translation stage (10) to drive the measured sample (9) to move, and make the laser focus on the measured sample (9) , obtain the CARS spectral information of this point by spectral detection unit (17). e) Separate processing of Rayleigh light signals can realize high spatial resolution three-dimensional scale tomography; separate processing of CARS spectral signals can obtain spectral images; simultaneous processing of Rayleigh signals and CARS spectral signals can achieve high spatial resolution of the map layer Analytical imaging, that is, to realize the integrated imaging and detection of the geometric position information and spectral information of the measured sample. 2. A kind of sub-pupil laser differential confocal CARS microspectral testing method according to claim 1, characterized in that the illumination pupil (7) and the collection pupil (8) can be circular, D-shaped or other shape. 3. a kind of split-pupil laser differential confocal CARS microspectral testing method according to right 1, is characterized in that, excitation beam comprises polarized light beams such as linearly polarized light, circularly polarized light, radially polarized light and is filtered by the pupil etc. The structured beam generated by the technology improves the signal-to-noise ratio of the system's spectral signal and the lateral resolution of the system. 4. a kind of sub-pupil laser differential confocal CARS microspectral testing method according to right 1, is characterized in that, by matching the filter of different spectral bands, selects the Stokes light of different spectral bands, can Spectral detection of different spectral bands is realized; wherein, the filtering bands of the band-pass filter (4) and the band-pass filter (12) are symmetrical about the central wavelength of the single-wavelength laser (2). 5. a kind of sub-pupil laser differential confocal CARS microspectral testing method according to right 1, is characterized in that, laser emission unit (1) can also add photonic crystal fiber (35) with single-wavelength laser (2) Spectral broadening is carried out. In addition, the spectrometer in the spectral detection unit is replaced by a photoelectric point detector (39), and the rotating polarizer (34) can realize the spectral scanning output, and then excite the CARS spectrum and obtain the CARS spectrum by the photoelectric point detector. Signal. 6. A differential confocal CARS microscopic spectrum testing device comprises: a spectrum excitation unit, a laser emission unit (1) positioned at the incident direction of the spectrum excitation unit, a dichroic unit (11) positioned at the exit direction of the spectrum excitation unit, a The spectral detection unit (17) in the transmission direction of the dichroic unit (11), the split-pupil laser differential confocal detection unit (18) located in the reflection direction of the dichroic unit (11), and the computer (40) controlling the entire system. The laser emission unit (1) is composed of a single-wavelength pulse laser (2), a supercontinuum pulse laser (3), a bandpass filter (4) and a dichroic mirror (5); the spectrum excitation unit is composed of an illumination pupil ( 7), the collection pupil (8), the microscope objective lens (6), the sample to be tested (9) and the high-precision three-dimensional scanning translation platform (10); the dichroic unit (11) is the second dichroic mirror ( 11); Spectral detection unit (17) is made up of bandpass filter (12), the first converging mirror (13), the first pinhole (14), the second converging mirror (15) and spectrometer (16); The pupil laser differential confocal detection unit (18) is composed of a third converging mirror (19) and a light intensity collection system (20). 7. a kind of split-pupil laser differential confocal CARS microscopic spectroscopic testing device as claimed in claim 6, is characterized in that, light intensity collection system (20) can adopt double pinhole (27) and two quadrant detectors ( 28) Combined method to realize segmentation and detection of Airy disk. 8. A kind of split-pupil laser differential confocal CARS microscopic spectroscopic testing device as claimed in claim 6, is characterized in that, the light intensity collection system (20) can adopt CCD detector (29), passes through on CCD detection surface Set the position and size of the detection area to realize the segmentation detection of the Airy disc. 9. A kind of split-pupil laser differential confocal CARS microscopic spectroscopic testing device as claimed in claim 6, is characterized in that, the light intensity acquisition system (20) can adopt the conduction optical fiber (30,31), through the third On the focal plane of the converging mirror (19), two optical fibers are placed symmetrically with respect to the optical axis to realize split detection of the Airy disk. 10. A kind of split-pupil laser differential confocal CARS micro-spectroscopy testing device as claimed in claim 6, is characterized in that, by increasing the image magnification system (32), the Airy disk detected by the amplified light intensity acquisition system , to improve the acquisition accuracy of the split-pupil laser differential confocal measurement device.