CN107167457A - The confocal CARS micro-spectrometers method and device of transmission-type - Google Patents

Abstract

The invention belongs to the technical field of microspectral imaging detection, and relates to a transmission-type confocal CARS microspectral testing method and device. The core idea of the present invention is to integrate laser confocal microscopy technology and CARS spectral detection technology, and add a two-way spectroscopic unit in the transmission confocal microstructure to perform non-destructive separation of Rayleigh light and CARS light, wherein CARS light performs spectral detection, and Rayleigh light performs geometric detection. position. The present invention utilizes the characteristic that the vertex of the confocal curve corresponds to the focus position precisely, accurately captures and locates the focus position of the excitation spot, realizes high-precision geometric detection and high-spatial-resolution spectral detection, and constitutes a high-spatial-resolution micro-area of the sample. Method and apparatus for spectral detection. By combining CARS microscopy technology, the excited Raman scattered light carrying transparent sample information is much stronger than traditional excited Raman light, and the excitation time is short, which provides the possibility for rapid detection of biological samples and transparent materials. The invention has the advantages of accurate positioning, high spatial resolution, high spectral detection sensitivity and controllable measurement focus spot size, and has wide application prospects in the fields of biomedicine, transparent material detection and the like.

Description

Transmission confocal CARS microspectral testing method and device

technical field

The invention belongs to the technical field of microspectral imaging, and relates to a transmission-type confocal CARS microspectral testing method and device, which can be used to quickly detect the geometric shape of a transparent sample to be tested and obtain a micro-area CARS spectrum to achieve high spatial resolution. Tomographic imaging.

technical background

Due to the characteristics of Raman scattering itself, the traditional confocal Raman microscopy technique results in extremely weak scattering spectral signals. 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 the frequency between Ep and Es When the difference matches the vibrational frequency of the Raman-active molecule, the excited signal is resonantly enhanced.

Most of the existing CARS microscopy techniques use two single-wavelength lasers for spectral excitation, so only spectral information of a specific spectrum can be obtained, and the system’s fixed-focus capability is not emphasized, 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. As a result, the ability of the CARS microscopic system to detect micro-region spectra is greatly limited, and its application in finer micro-region spectral testing and analysis occasions is restricted.

In addition, most spectroscopic detection techniques and devices are based on reflective structures, which are not effective in detecting transparent materials. Based on the above situation, the present invention proposes high-precision detection of the Rayleigh light scattered in the sample collected by the system, which is 10 ³ to 10 ⁶ times stronger than the Raman scattered light of the sample, for the measurement of transparent samples, so that it can be organically integrated with the spectral detection system. Simultaneous detection of spatial position information and spectral information is performed to realize confocal CARS atlas imaging and detection with high spatial resolution and high spectral resolution.

The core idea of the patent of the present invention is to propose a transmission confocal CARS microspectral measurement method and device based on the requirement of detecting transparent samples. Among them, single-wavelength pulsed lasers and supercontinuum pulsed lasers are selected as excitation light sources to expand the range of excitation spectra and increase the intensity of spectral excitation; combine the confocal microstructure with the CARS spectral structure, use the "maximum point" of the confocal response curve and the The focus position of the micro-objective lens accurately corresponds to this characteristic, precise focusing, and high spatial resolution; after precise focusing, spectral detection is performed to obtain the best spectral resolution capability; spectral information and geometric information are fused to achieve tomographic detection and measurement .

Contents of the invention

The purpose of the present invention is to overcome the deficiencies of the prior art, and propose a transmission confocal CARS microspectral testing method and device for testing transparent samples.

The present invention is achieved through the following technical solutions. A laser confocal CARS spectrum testing method, comprising the following steps:

a) In the laser emitting unit (1), the supercontinuum laser is emitted by the supercontinuum laser (3), after passing through a bandpass filter (4) to obtain the Stokes light of the required spectrum, passing through a two The chromatic mirror (5) is mixed with the single-wavelength laser light emitted by the single-wavelength laser (2) to form a mixed beam (consistent in frequency, consistent in time, and overlapping in space) and enters the spectrum excitation unit (6);

b) In the spectrum excitation unit (6), the mixed light beam is reflected by the first mirror (7) and passes through the microscope objective lens (8), focusing on the measured sample (10) fixed on the three-dimensional scanning platform (9), Excite Rayleigh light and anti-Stokes (CARS) light carrying the spectral characteristics of the measured sample (10), and the mixed beam formed after passing through the sample includes Rayleigh light, Stokes light, and CARS light;

c) The mixed beam is collected by the microscope objective lens (11), and is divided into two beams after passing through the dichroic mirror (12), wherein the beam containing CARS light enters the spectrum detection unit (13), and the other beam containing Rayleigh light enters the common Focus detection system (19); in spectrum detection unit (13), the light beam that comprises CARS light first passes through band-pass filter (14), filters the non-CARS interfering light in the light beam, then passes through the first condenser lens (15) Converge into the spectrometer (18) to obtain CARS spectral information; another beam carrying Rayleigh light passes through the third condenser lens (20) and passes through the second pinhole (21) at the focal point to filter out stray light and then is detected by the photodetector (22) Detection to obtain confocal signals. When the three-dimensional translation stage (9) moves in the Z direction, the confocal signal intensity changes accordingly, and the confocal response curve (24) is obtained, and the "maximum value" point of the confocal response curve is used to accurately correspond to the focal position of the microscope objective lens (8). characteristics, the Z-direction geometric position of the tested sample is obtained, combined with the lateral position of the three-dimensional translation stage (9), the measured sample geometric shape I(x, y, z) is obtained.

d) After the microscopic objective lens is precisely focused by the three-dimensional translation stage (9), the spectral information I(λ) of the current measurement point is obtained through the spectral detection unit (13); the system can obtain high spatial The resolved tomographic image of the micro-region map realizes transmission confocal CARS microspectral detection.

Particularly, in the method of the present invention, in the laser emission unit, the single-wavelength pulsed laser (2) and the supercontinuum pulsed laser (3) are pulsed lasers with the same repetition frequency.

In particular, by matching filters of different bands and selecting Stokes light of different bands, spectral detection of different bands is realized.

In particular, in the method of the present invention, the implementation of the laser emitting unit (1) also includes using a single-wavelength pulse laser to add a photonic crystal fiber (26) to expand the supercontinuum and then mix and output it with the single-wavelength laser.

In particular, in the method of the present invention, the way of exciting the CARS light also includes the way of reverse excitation.

A transmission-type confocal CARS microspectral testing device, comprising the following five parts: a laser emission unit (1), a spectrum excitation unit (6), a spectrum detection unit (13), a confocal detection unit (19), a computer ( 33). 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 consists of a first reflector (7), composed of the first microscopic objective lens (8), three-dimensional translation stage (9), measured sample (10), second microscopic objective lens (11), dichroic mirror (12); spectral detection unit (13 ) is made up of band-pass filter (14), the first condenser lens (15), the first pinhole (16), the second condenser lens (17), spectrometer (18); Confocal detection unit (19) is made up of the 3rd condenser lens (20), the first pinhole (21), and the first photodetector (22).

In the device of the present invention, the spectral detection unit (13) is located in the transmission direction of the dichroic mirror (12), the confocal detection unit (19) is located in the reflection direction of the dichroic mirror (12), and the computer (33) and the three-dimensional translation stage ( 9), the spectral detection unit (13) and the confocal detection unit (19) are connected to fuse and process spectral data and position information.

In the device of the present invention, the spectral detection unit (13) can also be an ordinary spectral detection unit composed of a bandpass filter (14), a first condenser mirror (15), and a spectrometer (18).

In the device of the present invention, the CARS optical excitation mode includes: when the light emitted by the single-wavelength laser (2) is used as pump light and probe light, the continuous spectrum laser emitted by the continuous spectrum laser (3) is used as Stokes light; When the light emitted by the single-wavelength laser (2) is used as Stokes light and probe light, the continuous spectrum laser emitted by the continuous spectrum laser (3) is used as pumping light.

In the device of the present invention, a polarization modulator is added between the laser emitting unit (1) and the spectral excitation unit (6) to form a polarized beam to improve the signal-to-noise ratio of the spectral excitation signal, or a pupil filter (32) is added to generate a structured beam to improve geometric resolution;

Beneficial effect

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

1. The present invention integrates transmission confocal microscopy technology and CARS spectral detection technology, realizes high-precision geometric measurement and fixed focus through confocal microscopy technology, and performs CARS spectrum excitation and measurement on samples after fixed focus, greatly improving the microscopic efficiency of existing CARS spectral microscopes. Area spectral detection capability;

2. The present invention adopts double symmetrical microscope objective lens, which can be switched synchronously by computer, which improves the operability of the instrument;

3. The two lasers used in the present invention are narrow-pulse femtosecond lasers with the same start time and repetition frequency, with stable optical path and high excitation efficiency.

4. The present invention can realize micro-area three-dimensional shape measurement and imaging, and can also realize micro-area spectral measurement and imaging, and can also combine the two to achieve high spatial resolution tomography;

5. The present invention adopts a transmission structure, which makes up for the shortcomings of the reflective spectral measurement method in measuring transparent sample signal strength is weak and difficult to collect;

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 energy level relationship between CARS light, pump light, and Stokes light;

Fig. 4 is a schematic diagram of a transmission confocal CARS microspectral measurement method;

5 is a schematic diagram of a transmission confocal CARS microspectral measurement method for improved spectral detection;

Figure 6 is a schematic diagram of multi-layer sample measurement

Fig. 7 is a schematic diagram of a transmission confocal CARS microspectral measurement method realized by a single laser;

8 is a schematic diagram of a transmission confocal CARS microspectral measurement method for time-domain scanning;

Fig. 9 is a schematic diagram of a transmission confocal CARS microspectral testing method for reverse excitation;

10 is a schematic diagram of a transmission confocal CARS microspectral testing method with a pupil filter added;

Fig. 11 is a schematic diagram of a transmission confocal CARS microspectral testing device, which is a diagram for an embodiment;

Among them, 1-laser emission unit, 2-single-wavelength laser source, 3-supercontinuum laser source, 4-bandpass filter, 5-dichroic mirror, 6-spectral excitation unit, 7-first mirror , 8-first microscope objective lens, 9-three-dimensional translation stage, 10-measured sample, 11-second microscope objective lens, 12-dichroic mirror, 13-spectral detection unit, 14-bandpass filter, 15-first condenser, 16-first pinhole, 17-second condenser, 18-spectrometer, 19-confocal detection unit, 20-third condenser, 21-second pinhole, 22-photodetector, 23 -focusing spot, 24-multilayer confocal curve, 25-polarization beam splitter, 26-photonic crystal fiber, 27-second mirror, 28-optical delayer, 29-third mirror, 30-polarizer, 31-photoelectric point detector, 32-pupil filter, 33-computer;

detailed description

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

As shown in Figure 4, the laser emitting unit (1) produces a spatially coincident, time-consistent mixed beam (Stokes light and pump light), which enters the spectral excitation unit (6); in the spectral excitation unit (6), The light beam is reflected by the first reflector (7) to the microscope objective lens (8), and is tightly focused on the measured sample (10) by the high-magnification microscope objective lens (8), which excites Rayleigh light and CARS light; CARS light, Rayleigh light and residual Stokes light are collected by the second microscopic objective lens (11), and split by the dichroic mirror (12), wherein CARS light and Stokes light enter the spectrum detection unit (13) Spectral detection is carried out, and Rayleigh light enters the confocal detection unit (19) for geometric position measurement;

Add the first pinhole (16) to filter stray light in the spectral detection unit (13) and improve the signal-to-noise ratio of spectral detection, which constitutes Fig. 5;

Fig. 6 is the schematic diagram of detecting transparent multi-layer samples by this method, the three-dimensional translation stage (9) moves along the Z direction, makes the light spot (23) size of the microscopic objective lens (8) focus on the multi-layer sample (10) to be tested change, corresponding The CARS spectral signal and Rayleigh signal intensity collected by the spectral detection unit and the confocal detection unit change, and the Z-direction position and the confocal signal intensity change constitute the confocal curve (24). When the microscopic objective lens (8) focuses on different layers , the multilayer confocal curve (24) is obtained, and the positions of multiple peak points of the multilayer confocal curve (24) are obtained, so as to realize the layered focus detection of the tested sample (10);

In Fig. 4, the laser emitting unit composed of double lasers is replaced with a combined structure of a single laser and a photonic crystal fiber, which constitutes Fig. 7; in Fig. 7, a single-wavelength pulsed laser (2) emits a single-wavelength pulsed laser, which passes through a polarization beam splitter prism (25) light splitting, half of the light is carried out supercontinuum expansion to obtain supercontinuum laser through photonic crystal fiber (26), after selecting the laser that needs specific wavelength by band-pass filter (4), with the other half single-wavelength laser in two The dichroic mirror (5) overlaps, and the optical delayer (28) is adjusted to ensure that the two laser beams overlap in time, so as to meet the requirements of spatial overlap and time consistency of CARS light excitation.

Add a polarizer (30) between the polarization beam splitter prism (25) and the photonic crystal fiber (26) in Fig. 7, and change the spectrometer in the spectral detection unit (13) into a photoelectric point detector with rapid response and sensitive detection (31), promptly constitute Fig. 8; Like this by rotating polarizer (30), change the laser polarization state of incident photonic crystal fiber (26), make the wavelength conversion of the continuous spectrum laser of output, realize the time-domain scanning output of spectrum, combine The photoelectric point detector (31) can realize the time-domain scanning detection of the CARS spectrum, which is one of the methods for exciting and detecting the CARS spectrum in the method of the present invention.

In the method of the present invention, in addition to adopting the method of excitation in the same direction and detection in the opposite direction to excite the CARS light, the method of excitation in the opposite direction and detection in the opposite direction can also be used. As shown in Figure 9, the single-wavelength laser light emitted by the single-wavelength laser (2) is reflected by the dichroic mirror (5) and converged on the sample through the second microscope objective lens (11), while the light emitted by the supercontinuum laser (3) The continuum laser beam is reflected by the reflector (7) and converged on the sample (10) through the first microscope objective lens (8); the positions of the two beams of light converged on the sample are the same, ensuring that the timing of the two pulsed laser beams arriving at the sample position is consistent, and the excitation The Rayleigh light and the CARS light carrying the spectral characteristics of the sample to be measured respectively enter the spectral detection unit (13) and the confocal detection unit (19) for geometric position detection.

Based on the basic implementation method of the present invention, a pupil filter (32) is added between the laser emitting unit (1) and the spectral excitation unit (6), which constitutes Fig. 10; at this time, by introducing structured light beams, the spatial resolution of the system is improved can be improved;

Example

In this embodiment, a femtosecond laser with a wavelength of 800 nm is used as the pump light source and a probe light source, and a supercontinuum (400-2000 nm) picosecond laser with the same repetition frequency is used as the Stokes light source.

As shown in Figure 11, the transmission-type confocal CARS micro-spectroscopy test device, the test steps are as follows:

In the laser emitting unit (1), the single-wavelength pulsed laser (2) and the supercontinuum pulsed laser (3) simultaneously emit pulsed lasers, and the supercontinuum laser selects a suitable Stokes through a bandpass filter (4). Si light (810-950nm) and single-wavelength laser (900nm) converge at the dichroic mirror (5) to ensure that after the two laser beams merge, the space coincides (the single-wavelength laser envelopes the continuum laser) and the time is consistent (two The optical path of the laser beam reaching the beam splitter is the same);

The mixed light beam is tightly focused on the sample to be tested (10) through the high-magnification microscope objective lens (8). At this time, the phase matching condition is met, and the Rayleigh light and the wavelength range of 690-790nm carrying the spectral characteristics of the sample to be tested are excited. Anti-Stokes light (CARS). The CARS light enters the spectral detection unit (13) for detection, and the Rayleigh light enters the confocal detection unit (19) for geometric position detection.

In the standard measurement mode, the computer controls the three-dimensional translation platform (9) to move in the X-Y direction to realize horizontal scanning. When measuring a single point, the computer controls the three-dimensional translation platform to move in the Z direction. At this time, the light spot (23) on the sample (10) to be measured When the size changes, the confocal signal intensity I(z) obtained by the corresponding confocal detection unit (19) changes, and the measured point is obtained according to the maximum point of the confocal intensity response curve (24) corresponding to the focal position of the microscopic objective lens (8). Geometric position information I (x, y, z); At this time, the measured sample (10) is moved to the focus position of the objective lens by the three-dimensional translation stage (9), and the spectral signal I at this time is measured by the spectral detection unit (13) (λ), combined with the position of the three-dimensional translation platform to obtain spectral information I(x, y, λ), the geometric position information I(x, y, z) and spectral information I(x, y, λ) are fused by computer (33) , to obtain spectral image information I(x,y,z,λ) with high spatial resolution. The connection between the three is as follows:

micromap tomography

By fusing the geometric position information I(x, y, z) and the spectral information I(x, y, λ), the micro-region map imaging I(x, y, z, λ) can be realized.

As shown in Figure 11, the transmission type confocal CARS microscopic spectrum testing device comprises a laser emission unit 1, a spectrum excitation unit (6), a spectrum detection unit (13), a confocal detection unit (19), and a computer (33); wherein The laser emission unit (1) comprises a single-wavelength laser light source (2), a supercontinuum laser light source (3), a bandpass filter (4), a dichroic mirror (5); the spectrum excitation unit (6) comprises a first Mirror (7), first microscopic objective lens (8), three-dimensional translation stage (9), measured sample (10), second microscopic objective lens (11), dichroic mirror (12); spectral detection unit ( 13) comprise bandpass filter (14), the first condenser lens (15), the first pinhole (16), the second condenser lens (17), spectrometer (18); Confocal detection unit (19) comprises the 3rd condenser lens (20), the second pinhole (21), photodetector (22);

Wherein, the spectral excitation unit (1) is located in the incident direction of the mirror (7) in the spectral excitation unit (6), the spectral detection unit is located in the transmission direction of the dichroic mirror (12) in the spectral excitation unit (6), and the confocal detection The unit (19) is located in the reflection direction of the dichroic mirror (12);

In the whole system, the single-wavelength laser source (2), the supercontinuum laser source (3), the three-dimensional translation stage (9), the spectrometer (18), and the photodetector (22) are all controlled by the computer (33), and the system is obtained The three-dimensional position information and spectral information of the computer (33) are also fused.

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 transmission confocal CARS microspectral testing method and device, which mainly carry out spectrum imaging to transparent samples, characterized in that: a) In the laser emitting unit (1), the supercontinuum laser is emitted by the supercontinuum laser (3), after passing through a bandpass filter (4) to obtain the Stokes light of the required spectrum, passing through a two The chromatic mirror (5) is mixed with the single-wavelength laser light emitted by the single-wavelength laser (2) to form a mixed beam (consistent in frequency, consistent in time, and overlapping in space) and enters the spectrum excitation unit (6); b) In the spectrum excitation unit (6), the mixed light beam is reflected by the first mirror (7) and passes through the microscope objective lens (8), focusing on the measured sample (10) fixed on the three-dimensional scanning platform (9), Excite Rayleigh light and anti-Stokes (CARS) light carrying the spectral characteristics of the measured sample (10), and the mixed beam formed after passing through the sample includes Rayleigh light, Stokes light, and CARS light; c) The mixed beam is collected by the microscope objective lens (11), and is divided into two beams after passing through the dichroic mirror (12), wherein the beam containing CARS light enters the spectrum detection unit (13), and the other beam containing Rayleigh light enters the common Focus detection system (19); In spectrum detection unit (13), the light beam that comprises CARS light passes through band-pass filter (14) earlier, filters the non-CARS interfering light in the light beam, then passes through first converging mirror (15) ) converges into the spectrometer (18) to obtain CARS spectrum information; another beam carrying Rayleigh light passes through the third converging mirror (20) and passes through the second pinhole (21) at the focal point to filter out stray light and then is photoelectrically The detector (22) detects to obtain confocal signals. When the three-dimensional translation stage (9) moves in the Z direction, the confocal signal intensity changes accordingly, and the confocal response curve (24) is obtained, and the "maximum value" point of the confocal response curve is used to accurately correspond to the focal position of the microscope objective lens (8). characteristics, the Z-direction geometric position of the tested sample is obtained, combined with the lateral position of the three-dimensional translation stage (9), the measured sample geometric shape I(x, y, z) is obtained. d) After the microscopic objective lens is precisely focused by the three-dimensional translation stage (9), the spectral information I(λ) of the current measurement point is obtained through the spectral detection unit (13); the system can obtain high spatial The resolved tomographic image of the micro-region map realizes transmission confocal CARS microspectral detection. 2. a kind of transmissive confocal CARS microspectral testing method according to right 1, is characterized in that, in the laser emission unit (1), single-wavelength pulsed laser (2) and supercontinuum pulsed laser (3) are A pulsed laser with a consistent repetition rate. 3. A kind of transmission type confocal CARS microspectral testing method according to right 1, it is characterized in that, by matching the filter of different spectral bands, select the Stokes light of different spectral bands, can realize different spectral Segment spectral detection. 4. according to a kind of transmission type confocal CARS micro-spectroscopy testing method according to right 1, it is characterized in that, the realization mode of laser emitting unit (1) of the present invention also comprises adopting a single-wavelength pulsed laser to add photonic crystal fiber (26 ) are mixed with single-wavelength laser to output after supercontinuum expansion. 5. A transmission-type confocal CARS microspectral testing method according to claim 1, characterized in that the excitation of CARS light in the present invention also includes a reverse excitation method. 6. A transmission-type confocal CARS microscopic spectrum testing device, comprising the following five parts: a laser emission unit (1), a spectrum excitation unit (6), a spectrum detection unit (13), a confocal detection unit (19), computer (33). 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 consists of a first reflector (7), composed of the first microscopic objective lens (8), three-dimensional translation stage (9), measured sample (10), second microscopic objective lens (11), dichroic mirror (12); spectral detection unit (13 ) consists of a bandpass filter (14), the first converging lens (15), the first pinhole (16), the second converging lens (17), and a spectrometer (18); the confocal detection unit (19) consists of the first It consists of three converging mirrors (20), a first pinhole (21), and a first photodetector (22). 7. A kind of transmission type confocal CARS micro-spectrum testing device as claimed in claim 6, is characterized in that, spectral detection unit (13) is positioned at dichroic mirror (12) transmission direction, confocal detection unit (19) Located in the reflection direction of the dichroic mirror (12), the computer (33) is connected with the three-dimensional translation stage (9), the spectral detection unit (13), and the confocal detection unit (19) for fusion and processing of spectral data and position information. 8. A kind of transmission type confocal CARS micro-spectroscopy testing device as claimed in claim 6, is characterized in that, spectral detection unit can also be made of band-pass filter (14), the first converging lens (15), A common spectral detection unit formed by a spectrometer (18). 9. A kind of transmission type confocal CARS micro-spectroscopy testing device as claimed in claim 6, the CARS optical excitation mode of the device of the present invention comprises: when the light that single-wavelength laser (2) sends is used as pumping light and probe light , the continuum laser that continuum laser (3) sends is as Stokes light; Laser as pump light. 10. A kind of transmission-type confocal CARS micro-spectroscopy testing device as claimed in claim 6, between the laser emission unit (1) and the spectrum excitation unit (6), add a polarization modulator to form a polarized light beam to improve the spectrum excitation signal-to-noise ratio, or add a pupil filter (32) to generate a structured beam to improve geometric resolution.