CN107192702B - Spectroscopic pupil laser confocal CARS (coherent anti-Raman scattering) microspectroscopy testing method and device - Google Patents

Spectroscopic pupil laser confocal CARS (coherent anti-Raman scattering) microspectroscopy testing method and device Download PDF

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CN107192702B
CN107192702B CN201710366654.XA CN201710366654A CN107192702B CN 107192702 B CN107192702 B CN 107192702B CN 201710366654 A CN201710366654 A CN 201710366654A CN 107192702 B CN107192702 B CN 107192702B
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cars
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pupil
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spectrum
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CN107192702A (en
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邱丽荣
吴寒旭
赵维谦
王允
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Beijing University of Technology
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/65—Raman scattering
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/6402—Atomic fluorescence; Laser induced fluorescence
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/645—Specially adapted constructive features of fluorimeters
    • G01N21/6456—Spatial resolved fluorescence measurements; Imaging
    • G01N21/6458—Fluorescence microscopy
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/65—Raman scattering
    • G01N2021/653—Coherent methods [CARS]
    • G01N2021/655—Stimulated Raman

Abstract

The invention belongs to the technical field of microspectroscopic imaging detection, and relates to a spectroscopic pupil laser confocal CARS microspectroscopic testing method and device. The core idea of the invention is to fuse the spectroscopic pupil laser confocal microscopy technology and the CARS spectrum detection technology, and to perform nondestructive separation on Rayleigh light and CARS light by adopting a two-way spectroscopic system, wherein the CARS light is subjected to spectrum detection, and the Rayleigh light is subjected to geometric positioning. The invention utilizes the characteristic that the vertex of the pupil-splitting laser confocal curve and the focal position are accurately corresponding to each other, accurately captures and positions the focal position of an excitation light spot, realizes high-precision geometric detection and high-spatial-resolution spectral detection, and forms a method and a device capable of realizing the high-spatial-resolution spectral detection of a sample micro-area. By combining the CARS microscopic technology, the excited Raman scattering light carrying the sample information is far stronger than the traditional spontaneous Raman light, and the excitation time is short, thereby providing possibility for rapidly detecting biological samples and chemical materials. The invention has the advantages of accurate positioning, high spatial resolution, high spectral detection sensitivity, controllable size of a measured focusing light spot and the like, and has wide application prospect in the fields of biomedicine, material detection and the like.

Description

Spectroscopic pupil laser confocal CARS (coherent anti-Raman scattering) microspectroscopy testing method and device
Technical Field
The invention belongs to the technical field of microspectroscopic imaging, and relates to a pupil-splitting laser confocal CARS microspectroscopic testing method and device, which can be used for rapidly detecting micro-region anti-Stokes scattering (CARS) spectrums of various samples, can realize geometric imaging and detection with high spatial resolution, and can obtain a map-in-one image with high spatial resolution.
Technical Field
Optical microscopes are widely used in the biomedical and material science fields, and with the rapid development of modern science, the requirements for microscopic imaging are also shifted from structural imaging to functional imaging. In 1990, the successful application of confocal Raman spectrum microscopy greatly improves the possibility of exploring the specific tissue components and the morphology of a micro object. The confocal microscopic technology and the Raman spectrum technology are combined, the confocal microscopic device has the high-resolution tomography characteristic of confocal microscopy, has the capabilities of nondestructive detection and spectral analysis, becomes an important technical means for material structure measurement and analysis, and is widely applied to the fields of physics, chemistry, biomedicine, material science, petrochemical industry, food, medicines, criminal investigation and the like.
The traditional spontaneous Raman scattering imaging technology has extremely weak emission signals due to the characteristics of Raman scattering, and even if high-intensity laser is used for excitation, a long action time is still needed for obtaining a spectral image with good contrast. This long-term effect limits the application of raman microscopy in the biological field. A coherent anti-stokes raman scattering (CARS) process based on the coherent raman effect can enhance the raman signal to a large extent, thereby enabling fast detection. The coherent raman effect is that molecules are locked on a vibration energy level through excited light, and the intensity of a vibration signal generated by the method is in a nonlinear relation with the intensity of the excited light, so that a strong signal can be generated, and the coherent raman effect is also called coherent nonlinear raman spectrum. The method has the advantages of strong energy conversion efficiency, short exposure time, less damage to the sample, and easy separation from stray light due to certain directivity of scattering.
Coherent anti-stokes raman scattering (CARS) generation is a third-order nonlinear optical process that requires a pump light, a stokes light and a probe light. In general, in order to reduce the number of light sources and simplify the process, the probe light is replaced by the pump light, and the relationship between them is shown in fig. 2 when the pump light (w) isp) And Stokes light (w)s) When the frequency difference of the Raman active molecule is matched with the vibration frequency of the Raman active molecule, CARS light w is excitedasWherein w isas＝2wp-ws. The generation process of CARS light comprises the interaction process of a specific vibrational mode of raman active molecules and an incident optical field that causes vibrational transition of the molecules from a ground state to an excited state, and its energy level diagram is shown in fig. 3. FIG. 3(a) shows the contribution of Raman resonance and non-resonance single photon enhancement to the CARS process, and FIG. 3(b) shows the contribution of Raman resonance and non-resonance two-photon enhancement to the CARS process; when w ispAnd wsWhen the frequency difference between the two signals is matched with the vibration frequency of the raman active molecule, the excited signal is resonantly enhanced, and the non-resonant part is also enhanced due to the electron transition response, so that to obtain a better CARS signal, the non-resonant background signal needs to be suppressed as much as possible, and the common method is the polarization CARS (P-CARS) method.
The principle of P-CARS is shown in FIG. 4, where the light source 1 emits light with a frequency wpAfter polarization, the Stokes light passes through a quarter-wave plate and a half-wave plate and then has a frequency w emitted by the light source 2sThe pump light (probe light) is converged, is emitted to a water immersion microscope objective lens by a reflector after passing through a two-way beam splitter, is focused on a sample, excites CARS light with spectral characteristics and then transmits the CARS light into a signal acquisition system; the signal is collected by an oil-immersed microscope objective, a non-resonance background is filtered by a polaroid, then the interference of other spectral bands is filtered by an optical filter, and the signal is collected by an avalanche photodiode, so that the spectral signal of a specific frequency spectrum is obtained.
The P-CARS can suppress the interference of non-resonance signals and excitation light to a large extent, but its wide use is greatly limited because it uses two single-wavelength lasers and can only obtain spectral information of a specific spectrum.
Conventional CARS microscopy does not emphasize the fixed focus capability of the system, resulting in the actual spectral detection position often being at an out-of-focus position. Even if the light can still excite the Raman spectrum of the sample at the out-of-focus position and be detected by a spectrometer behind the pinhole, the intensity cannot reasonably represent the correct spectral signal intensity at the point. In CARS microscopy systems, the best spatial resolution and the best spectral detection capability can only be obtained if the system is precisely focused.
Due to the reasons, the capability of the CARS microscopic system for detecting the spectrum of the micro-area is limited, and the application of the CARS microscopic system in the occasion of testing and analyzing the spectrum of the finer micro-area is restricted. Based on the above situation, the present invention proposes that the surface of the sample collected by the system is scattered more strongly than the Raman scattered light 10 of the sample3～106And the multiplied Rayleigh light is subjected to high-precision detection, so that the Rayleigh light is organically fused with the spectrum detection unit, and the spatial position information and the spectrum information are synchronously detected, so that the high-spatial-resolution and high-spectral-resolution pupil laser confocal CARS microscopic atlas imaging and detection are realized.
The core idea of the invention is to select a super-continuum spectrum pulse laser and a single-wavelength pulse laser as excitation light sources, expand the excitation spectrum range and improve the spectrum excitation intensity; combining a pupil laser confocal microstructure with a CARS spectral structure, and accurately focusing by utilizing the characteristic that a maximum value point of a pupil laser confocal response curve accurately corresponds to the vertex position (the focus position of a microscope objective) of a sample to be detected so as to realize high spatial resolution; after the focus is accurately determined, the spectrum detection is carried out to obtain the optimal spectrum resolution capability.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provides a spectral pupil laser confocal CARS microspectroscopy testing method and a device thereof with high spatial resolution.
The invention is realized by the following technical scheme. A pupil splitting laser confocal CARS micro-spectrum testing method comprises the following steps:
a) in the laser emission unit, the supercontinuum laser emits supercontinuum laser, and the supercontinuum laser passes through a band-pass filter and is coupled with the single-wavelength laser emitted by the single-wavelength laser through a first dichroic mirror to form mixed light beams; the mixed light beam is converged on a measured sample after passing through an illumination pupil and a measuring objective lens, and the CARS light carrying the spectral characteristics of the measured sample is excited, and simultaneously rayleigh light is reflected; the CARS light and the Rayleigh light pass through the measuring objective lens and the collecting pupil and are divided into two beams by the second dichroic mirror, wherein the light beam containing the CARS light enters the spectrum detection unit, and the other light beam containing the Rayleigh light enters the spectral pupil laser confocal detection unit; in the spectrum detection unit, a light beam containing CARS light firstly passes through a band-pass filter to filter non-CARS interference light in the light beam, then is converged through a first pinhole by a first converging lens, and is converged into a spectrometer by a second converging lens after ambient light is filtered to obtain CARS spectrum information; in the spectral pupil laser confocal detection unit, a light beam containing Rayleigh light passes through a third converging lens and is subjected to focal spot segmentation detection by a light intensity acquisition system to obtain a spectral pupil laser confocal intensity signal I (x, y, z);
b) by utilizing the characteristic that a spectral pupil laser confocal response curve 'maximum point' accurately corresponds to the focal position of a measuring microscope objective, spectral information I (x, y, lambda) is obtained by measuring after the focal position of an excitation light spot is accurately captured through the 'maximum point', so that high-spatial-resolution geometric detection and spectral detection are realized.
c) When processing the spectrum signal obtained by the spectrum detection unit receiving the CARS light, the system can perform spectrum detection (x, y, lambda); when the pupil laser confocal signal obtained by the pupil laser confocal detection unit which receives Rayleigh light is processed, the system can obtain a three-dimensional geometric shape (x, y, z); when the spectroscopic pupil laser confocal signal and the CARS signal are processed simultaneously, the system can perform high-spatial-resolution micro-area spectrum tomography (x, y, z and lambda), namely realize high-spatial-resolution imaging and detection of spectroscopic pupil laser confocal CARS microspectrum of integrating spectra of a detected sample.
In particular, in the method of the invention, the illumination pupil and the collection pupil may be circular, D-shaped or otherwise.
In particular, in the method of the present invention, the excitation light beam comprises polarized light beams such as linear polarized light, circular polarized light, radial polarized light and the like and structured light beams generated by pupil filtering technology, thereby improving the signal-to-noise ratio of the system spectral signals and the transverse resolution of the system.
Particularly, in the method, the Stokes light of different spectral bands is selected by matching optical filters of different spectral bands, so that spectral detection of different spectral bands can be realized; the band-pass filters and the filter bands of the band-pass filters are symmetrical about the central wavelength of the single-wavelength laser.
Particularly, in the method of the invention, the laser emission unit can also use a single-wavelength laser and photonic crystal fiber to perform spectrum broadening, in addition, a spectrometer in the spectrum detection unit is replaced by a photoelectric point detector, and the rotating polaroid can realize spectrum scanning output, so that a CARS spectrum is excited and a CARS spectrum signal is obtained by the detection of the photoelectric point detector;
the invention provides a spectroscopic pupil laser confocal CARS (coherent anti-interference device) microspectrum testing device which comprises a laser emission unit, a spectrum excitation unit positioned in the exit direction of the laser emission unit, a second dichroic mirror positioned in the exit direction of the spectrum excitation unit, a spectrum detection unit positioned in the transmission direction of the second dichroic mirror and a spectroscopic pupil laser confocal detection unit positioned in the reflection direction of the second dichroic mirror. The laser emission unit consists of a single-wavelength pulse laser, a super-continuum spectrum pulse laser, a band-pass filter and a first dichroic mirror; the spectrum excitation unit consists of a microscope objective, an illumination pupil, a collection pupil, a measured sample and a high-precision three-dimensional scanning translation stage; the spectrum detection unit consists of a band-pass filter, a first converging lens, a first pinhole, a second converging lens and a spectrometer; the spectral pupil laser confocal detection unit consists of a third converging lens and a light intensity acquisition system.
In the device, the light intensity acquisition system can realize the segmentation detection of the Airy spots by adopting a method of combining a pinhole with a light intensity point detector.
In the device, the light intensity acquisition system can adopt a CCD detector, and the segmentation detection of the Airy spots is realized by setting the position and the size of a detection area on a CCD detection surface.
In the device, the light intensity acquisition system can adopt optical fibers, and the segmentation detection of the Airy spots is realized by placing the conducting optical fibers on the optical axis on the focal plane of the third converging mirror.
In the device, the relay magnifying lens is added to magnify the Airy spots detected by the light intensity collecting system so as to improve the collecting precision of the spectral pupil laser confocal measuring device.
Advantageous effects
Compared with the prior art, the method of the invention has the following innovation points:
1. the invention integrates the pupil laser confocal microscopy technology and the CARS spectrum detection technology, and by the characteristic that the maximum point of the pupil laser confocal response curve corresponds to the focus of the high-precision microscope objective accurately, a focus sample is accurately fixed, the best CARS signal is obtained while the geometric position is obtained, the micro-area spectrum detection capability of the existing CARS spectrum microscope is greatly improved, and the optical path structure of the system is also greatly simplified, which is one of the innovation points different from the existing CARS spectrum detection technology;
2. the invention utilizes the dichroic beam splitting device to split Rayleigh light collected by the system and CARS light carrying sample information, then the Rayleigh light enters the beam splitting pupil laser confocal detection unit, and the CARS light enters the spectrum detection unit, thereby realizing reasonable utilization of light energy and improving the spectrum detection sensitivity of the system. The method is different from the current CARS spectrum detection technology in the second innovation point;
3. the invention adopts a method of dividing focal spots to obtain signals, and can match the reflectivity of different samples by changing the parameters of a micro area arranged on a detection focal plane of a light intensity acquisition system, thereby expanding the application field of the system; the matching of the measuring objective lenses with different NA values can be realized only by the software processing of a computer system, and the system does not need to be reset by any hardware, so that the universality of the instrument is favorably realized. The method is a third innovation point different from the conventional CARS spectrum detection technology;
4. the invention combines the spectroscopic pupil laser confocal microscope system and the CARS spectral imaging system in structure and function, can realize the tomography of the geometric parameters of the sample micro-area, can also realize the spectral detection of the sample micro-area, and can also combine the geometric position information and the spectral information obtained by the system to realize the spectral tomography, which is four innovative points different from the prior CARS spectral detection technology;
compared with the prior art, the method of the invention has the following remarkable advantages:
1. the precise focusing is realized by combining the spectroscopic pupil laser confocal detection technology, and the spatial resolution of CARS spectral detection is greatly improved;
2. the invention has three functions of geometric measurement, spectral test and spectrum tomography, and can meet the requirements of CARS spectral test to a greater extent.
3. The invention adopts a mode of combining broadband laser and single-wavelength laser to realize broadband CARS spectral measurement.
Drawings
FIG. 1 is a drawing of an abstract, which is a basic implementation diagram of the present invention;
FIG. 2 is a diagram of coherent anti-Stokes (CARS) light excitation principles;
FIG. 3 is a graph showing the relationship between CARS light and pump light and Stokes light
FIG. 4 is a conventional polarization detection microscopy path diagram;
FIG. 5 is a schematic diagram of a D-shaped pupil splitting laser confocal CARS microscopic test method;
FIG. 6 is a schematic diagram of a spectroscopic pupil laser confocal CARS microscopic test method incorporating a pupil filter;
FIG. 7 is a schematic diagram of a spectroscopic pupil laser confocal CARS microscopic test method using a pinhole and light intensity point detector;
FIG. 8 is a schematic diagram of a spectroscopic pupil laser confocal CARS microscopic test method using a CCD detector;
FIG. 9 is a schematic diagram of a spectroscopic pupil laser confocal CARS microscopic test method using optical fiber for detection;
FIG. 10 is a schematic diagram of a spectroscopic pupil laser confocal CARS microscopic testing device with a detection focal spot amplification system;
FIG. 11 is a schematic diagram of a spectroscopic pupil laser confocal CARS microscopic testing method for a single laser light source;
FIG. 12 is a schematic diagram of a high spatial resolution pupil-splitting laser confocal CARS microscopic test method and device, namely, an embodiment diagram.
The system comprises a laser emission unit 1, a single-wavelength laser light source 2, a supercontinuum laser light source 3, a band-pass filter 4, a first dichroic mirror 5, a measurement objective 6, a lighting pupil 7, a collection pupil 8, a sample 9 to be measured, a high-precision three-dimensional scanning translation table 10, a second dichroic mirror 11, a band-pass filter 12, a first converging lens 13, a first pinhole 14, a second converging lens 15, a spectrometer 16, a spectrum detection unit 17, a spectral confocal detection unit 18, a spectral confocal detection unit 19, a third converging lens 20, a light intensity collection system 21, a detection area 22, a spectral confocal curve 22, a pupil filter 23, a second pinhole 24, a spectral conversion unit 18, a spectral conversion unit, a spectral conversion, 25-light intensity point detector, 26-CCD detector, 27-conducting optical fiber, 28-relay magnifying lens, 29-polarization beam splitter prism, 30-polarizing plate, 31-photonic crystal fiber, 32-first reflector, 33-optical delay line, 34-second reflector, 35-photoelectric point detector and 36-computer;
Detailed Description
The present invention will be described in further detail with reference to the accompanying drawings and examples.
FIG. 1 is a schematic diagram of a spectroscopic pupil laser confocal CARS test method, wherein a laser emission unit (1) emits a mixed beam of single-wavelength pulse laser and continuous spectrum pulse laser (two pulse lasers are spatially coincident and time-coincident), the mixed beam passes through an illumination pupil (7) and a microscope objective (6) and enters the surface of a sample (9) to be tested at a certain angle α to excite CARS light and Rayleigh scattered light carrying spectral information of the sample (9), the Rayleigh light and the CARS light are recycled to a light path by the microscope objective (6) and a collection pupil (8) and are split by a second dichroic mirror (11), wherein the CARS light is transmitted into a spectrum detection unit (17) to perform spectral detection, the Rayleigh light is reflected into a spectroscopic pupil laser confocal detection unit (18) to perform light intensity detection, and the geometric position of a micro-area of the sample (9) to be tested is detected according to the position of a maximum point of a strength curve.
The circular illumination pupil (7) and the collection pupil (8) are replaced by other shapes, such as D-shapes, namely a D-shaped split pupil laser confocal CARS test method is formed, as shown in figure 5.
The spatial resolution of spectral detection is improved by adding a pupil filter (23), namely, a spectroscopic pupil laser confocal CARS test method with the pupil filter added is formed, as shown in figure 6.
The light intensity acquisition system (20) can realize the segmentation detection of the airy disk by adopting a method of combining a pinhole (24) and a light intensity point detector (25), as shown in fig. 7.
The light intensity acquisition system (20) can adopt a CCD detector (26), and realize the segmentation detection of the Airy spots by changing the parameters of a micro area arranged on a detection focal plane to match the reflectivity of different samples, thereby expanding the application field thereof, as shown in figure 8.
The light intensity acquisition system can adopt a conducting optical fiber (27), and the segmentation detection of the Airy spots is realized by placing an optical fiber on the optical axis at the focal plane of the third converging mirror (19), as shown in figure 9.
An amplifying system (28) can be added in the pupil laser confocal detection system to improve the acquisition accuracy of the pupil laser confocal measurement device, as shown in fig. 10.
FIG. 11 is a schematic diagram of a single-laser confocal CARS microscopic testing method, which is used for changing the double-laser input of a laser emission unit into single-laser input so as to reduce the cost; the single-wavelength pulse laser emits single-wavelength laser, the single-wavelength laser is split by a polarization splitting prism (29), a transmission part enters a photonic crystal fiber (31) through a polarizing film (30) to be subjected to spectral band broadening and is intercepted by a band-pass filter with specific required wavelength, a reflection part passes through a first reflector (32), an optical delay line (33) and a second reflector (34) and then is coupled with broadened continuous spectrum laser at a first dichroic mirror (5), mixed light beams with consistent space and time are output, and CARS spectrum excitation is carried out on a sample to be detected. The optical delay line (33) is used for ensuring the time sequence coincidence of the two laser beams. Further, a spectrometer (16) in the spectrum detection unit (17) is replaced by a photoelectric point detector (35), and the polarization state of a light beam is changed by the rotary polarizing film (30), so that a spectral line with continuously changed wavelength is output by the photonic crystal fiber (31) and is used as Stokes light, and further the broadband CARS spectrum measurement is realized.
Examples
In the embodiment, a picosecond laser with the wavelength of 1064nm is used as a pumping light source and a detection light source, a supercontinuum picosecond laser with the same repetition frequency is used, a band-pass filter of 1100-1300 nmm is added as a stokes light source, the mixture is emitted under the conditions of meeting space coincidence and time consistency, and is tightly focused on a sample through a high-power microscope objective, at the moment, the phase matching condition is met, and anti-stokes scattered light (CARS) with the wavelength range of 900-1030 nm is excited.
As shown in fig. 12, the spectroscopic pupil confocal CARS microspectroscopy testing apparatus comprises the following testing steps:
firstly, in a laser emission unit (1), continuous spectrum laser emitted by a super-continuous spectrum pulse laser (3) is filtered by a band-pass filter (4) to obtain broad band laser of 1100-1300 nm, and then the broad band laser and monochromatic laser emitted by a single-wavelength (1064nm) laser (2) are converged at a first dichroic mirror (5) to form a mixed light beam, wherein the repetition frequencies of the two beams of laser are consistent, the time for reaching a light splitting piece is consistent, and the beams can be completely superposed after being converged (pump light spots are completely enveloped in Stokes light spots); the mixed light beam is tightly focused on a tested sample (9) through an illumination pupil (7) and a microscope objective (8) to excite Rayleigh light and CARS light carrying the spectral characteristics of the tested sample (9). At this time, the sample can be three-dimensionally scanned by the high-precision three-dimensional scanning translation stage (10).
The light beam reflected back by the sample (9) to be measured contains Stokes light lambdasPump light lambda0Rayleigh light λ0CARS light lambdaas(ii) a Wherein, CARS light lambdaasAnd Stokes light λsPump light with 1064nm and Rayleigh light lambda enter a spectrum detection unit (17)0And the light is reflected by a second dichroic mirror (11) and enters a spectral pupil laser confocal detection unit (18). In a spectrum detection unit (17), only CARS light is reserved after light mixed by Stokes light and CARS light passes through a 900-1030 nm band-pass filter (12), then the light is converged through a first pinhole (14) by a first converging lens (13), ambient light interference is filtered, and then the light is converged into a spectrometer (16) by a second converging lens (15), so that a CARS spectrum I (x, y, lambda) is obtained by detection, wherein x and y represent the current measurement transverse position, and lambda is the wavelength of the CARS light excited by excitation light of a sample to be detected (9). In a spectroscopic pupil laser confocal detection unit (18), after being converged by a third converging lens (19), Rayleigh light is detected by a CCD detector (26) to obtain a confocal signal I (x, y, z), wherein x, y and z represent the three-dimensional position of a current measurement point;
under a standard measurement mode, a computer (36) controls a high-precision three-dimensional scanning translation table (10) to move to realize three-dimensional scanning, Z-direction tracking measurement is realized through a pupil splitting laser confocal detection unit (18), a pupil splitting laser confocal response curve (22) is obtained, and three-dimensional information I (x, y, Z) of a measured sample is obtained by combining the position of a three-dimensional scanning table according to the characteristic that the maximum point of the pupil splitting laser confocal intensity curve (22) accurately corresponds to the focal position of a measurement objective lens. After focusing, the spectrum detection unit (12) measures CARS spectrum information of the detected sample to obtain the CARS spectrum I (x, y, lambda) of the current measurement point.
I(x,y,z)+I(x,y,λ)＝I(x,y,z,λ)
The three-dimensional geometric information and the spectral information are combined, and the CARS spectral detection with high spatial resolution is realized.
Above, along laser outgoing direction, place laser emission unit (1) in proper order, place illumination pupil (7), measurement objective (6), by survey sample (9), three-dimensional scanning translation platform (10) in laser emission unit's emission direction, place collection pupil (8), second dichroic mirror (11) in measurement objective (6) reflection direction, place spectral detection unit (17) in second dichroic mirror (11) transmission direction, place the confocal detection unit of minute pupil (18) in second dichroic mirror (11) reflection direction. The laser emission unit (1) comprises a supercontinuum pulse laser (3), a band-pass filter (4), a first dichroic mirror (5) and a single-wavelength pulse laser (2); a band-pass filter (12), a first converging mirror (13), a first pinhole (14), a second converging mirror (15) and a spectrometer (16) are sequentially arranged in the spectrum detection unit (17); a third converging lens (19) and a CCD detector (26) are sequentially arranged in the spectroscopic pupil laser confocal detection unit (18); in the whole system, a single-wavelength pulse laser (2), a supercontinuum pulse laser (3), a high-precision three-dimensional scanning translation table (10), a spectrometer (16) and a CCD detector (26) are controlled by a computer (36), and three-dimensional position information and spectral information obtained by the system are fused by the computer (36).
While the invention has been described in connection with specific embodiments thereof, it will be understood that it is not intended to limit the scope of the invention, which is defined by the appended claims, any modifications that may be made based on the claims.

Claims (10)

1. A pupil splitting laser confocal CARS micro-spectrum testing method is characterized by comprising the following steps:
a) in the laser emission unit (1), a supercontinuum pulse laser (3) emits supercontinuum laser, and the supercontinuum laser passes through a band-pass filter (4) and is coupled with single-wavelength laser emitted by a single-wavelength laser (2) through a first dichroic mirror (5) to form mixed light beams; the mixed light beam is converged on a measured sample (9) through an illumination pupil (7) and a measurement objective lens (6), and CARS light carrying the spectral characteristics of the measured sample (9) is excited, and Rayleigh light is reflected; CARS light and Rayleigh light are collected by a measuring objective lens (6) and a collecting pupil (8), and then are divided into two beams by a second dichroic mirror (11), wherein the light beam containing the CARS light enters a spectrum detection unit (17), and the other light beam containing the Rayleigh light enters a confocal detection unit (18); in a spectrum detection unit (17), a light beam containing CARS light firstly passes through a band-pass filter (12) to filter non-CARS interference light in the light beam, then is converged by a first converging lens (13), a first pinhole (14) shields ambient light, the ambient light interference is reduced, and then the ambient light is converged by a second converging lens (15) to enter a spectrometer (16) to obtain CARS spectrum information; in the pupil laser confocal detection unit, a light beam containing Rayleigh light is converged by a third condenser lens (19) and then detected by a light intensity acquisition system (20) to obtain a pupil laser confocal intensity signal I (x, y, z);
b) the confocal signal intensity is changed along with the scanning of the high-precision three-dimensional translation table (10) to obtain a pupil-splitting laser confocal response curve, the focus position of an excitation light spot is accurately captured through a maximum point by utilizing the characteristic that the maximum point of the confocal response curve is accurately corresponding to the focus position of a measurement microscope objective, and then spectral information I (x, y, lambda) is obtained through measurement, so that the high-spatial-resolution geometric detection and spectral detection are realized;
c) when processing the spectrum signal obtained by the spectrum detection unit receiving the CARS light, the system can perform spectrum detection (x, y, lambda); when the pupil laser confocal signal obtained by the pupil laser confocal detection unit which receives Rayleigh light is processed, the system can obtain a three-dimensional geometric shape (x, y, z); when the bisection pupil laser confocal signal and the CARS signal are processed simultaneously, the system can perform high-spatial-resolution micro-area atlas tomography (x, y, z and lambda), namely, the high-spatial-resolution imaging and detection of the laser confocal CARS micro-spectrum integrating the atlas of the detected sample are realized.
2. The split-pupil laser confocal CARS microspectroscopy test method according to claim 1, characterized in that the illumination pupil (7) and the collection pupil (8) can be circular, D-shaped or other shapes.
3. The method for testing spectroscopic pupil-splitting laser confocal CARS method according to claim 1, wherein the excitation light beam comprises a linearly polarized light beam, a circularly polarized light beam, a radially polarized light beam and a structured light beam generated by pupil filtering technique, thereby improving the system spectral signal-to-noise ratio and the system lateral resolution.
4. The method for testing the spectroscopic of the pupil-splitting laser confocal CARS as claimed in claim 1, wherein the spectral detection of different spectral bands can be realized by matching optical filters of different spectral bands and selecting Stokes light of different spectral bands; wherein the filter 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. The method for testing the spectroscopic spectrum of the confocal CARS with the pupil laser as claimed in claim 1, wherein the laser emission unit (1) can be realized by performing spectral broadening by using a single-wavelength laser (2) and a photonic crystal fiber (31), and in addition, a spectrometer in the spectrum detection unit is replaced by a photoelectric spot detector (35), and a rotating polarizing plate (30) can realize spectral scanning output, so that the CARS spectrum is excited and a CARS spectrum signal is obtained by detecting the CARS spectrum by the photoelectric spot detector.
6. A pupil-splitting laser confocal CARS (coherent anti-interference Raman scattering) microspectrum testing device comprises a laser emitting unit (1), a spectrum excitation unit positioned in the emergent direction of the laser emitting unit (1), a second dichroic mirror (11) positioned in the emergent direction of the spectrum excitation unit, a spectrum detection unit (17) positioned in the transmission direction of the second dichroic mirror (11), and a pupil-splitting laser confocal detection unit (18) positioned in the reflection direction of the second dichroic mirror (11); the laser emission unit (1) consists of a single-wavelength pulse laser (2), a supercontinuum pulse laser (3), a band-pass filter (4) and a first dichroic mirror (5); the spectrum excitation unit consists of a measurement objective lens (6), an illumination pupil (7), a collection pupil (8), a measured sample (9) and a high-precision three-dimensional translation table (10); a second dichroic mirror (11), i.e. a second dichroic mirror (11); the spectrum detection unit (17) consists of a band-pass filter (12), a first converging mirror (13), a first pinhole (14), a second converging mirror (15) and a spectrometer (16); the spectroscopic pupil laser confocal detection unit (18) is composed of a third condenser lens (19) and a light intensity acquisition system (20).
7. The device for testing the pupil-splitting laser confocal CARS microspectrum according to claim 6, wherein the light intensity acquisition system (20) can realize the segmentation detection of the Airy spots by combining a pinhole (24) with a light intensity point detector (25).
8. The device for testing the pupil-splitting laser confocal CARS microspectrum according to claim 6, wherein the light intensity acquisition system (20) can adopt a CCD detector (26), and the position and the size of a detection area are set on a CCD detection surface to realize the segmentation detection of the Airy spots.
9. The device for testing spectroscopic pupil-splitting laser confocal CARS, according to claim 6, characterized in that the light intensity acquisition system (20) can use a conducting optical fiber (27) to realize the segmentation detection of Airy spots by placing the conducting optical fiber on the optical axis at the focal plane of the third converging mirror (19).
10. The device for testing spectroscopic measurement of confocal CARS of pupil laser according to claim 6, wherein the image magnifying system (28) is added to magnify the Airy spots detected by the light intensity collecting system to improve the collecting accuracy of the device for confocal measurement of pupil laser.
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