CN110579462B - Time-resolved broad-spectrum CARS spectral imaging device based on high-repetition-frequency femtosecond laser - Google Patents
Time-resolved broad-spectrum CARS spectral imaging device based on high-repetition-frequency femtosecond laser Download PDFInfo
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- 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
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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
- G01N21/01—Arrangements or apparatus for facilitating the optical investigation
- G01N2021/0106—General arrangement of respective parts
- G01N2021/0112—Apparatus in one mechanical, optical or electronic block
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- 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]
Abstract
The invention discloses a time-resolved broad-spectrum CARS spectral imaging device based on high-repetition-frequency femtosecond laser, which comprises a high-repetition-frequency femtosecond laser, a double-pulse laser generation module and a spectral imaging platform; the high repetition frequency femtosecond laser is used for generating fundamental frequency light with high repetition frequency and inputting the fundamental frequency light into the double-pulse laser generating module; the double-pulse laser generation module is used for generating coaxially transmitted fundamental frequency light and frequency shift light which are suitable for the time-resolved CARS imaging technology; the spectral imaging platform is used for realizing scanning type Raman spectral imaging on a sample to be detected. The invention adopts the femtosecond laser with wide spectrum as the pump light and the Stokes light at the same time, and can establish the coherence among a plurality of energy levels at one time, thereby realizing the hyperspectral imaging of various molecules at the same time; the coherent excitation process and the detection light excitation process which are realized by the pumping light and the Stokes light together are separated in time, and narrow spectrum light is adopted as the pumping light, so that the non-resonance background is effectively inhibited, and the detection sensitivity is greatly improved.
Description
Technical Field
The invention belongs to the technical field of optical imaging, and particularly relates to a time-resolved broad-spectrum CARS spectral imaging device based on high-repetition-frequency femtosecond laser.
Background
The optical imaging technology has wide application in the fields of material science, biomedicine and the like. At present, the conventional optical imaging technology still has a plurality of problems. For example, general optical imaging lacks chemical specificity, and cannot image the spatial distribution of a specific molecule in a sample, and the lack of chemical information will largely limit the application of imaging techniques; fluorescence labeling imaging, although chemically specific, requires labeling of specific molecules, i.e., complex pretreatment of the sample, which is not only time-consuming and labor-intensive, but also may change the characteristics of the sample, and for most molecules, methods for labeling are currently lacking; raman spectrum imaging can realize label-free molecular imaging, but is limited by extremely low efficiency of Raman scattering, and a signal with a high enough signal-to-noise ratio can be acquired only by long enough integration time, so that the imaging speed is greatly limited; in the CARS (coherent anti-Stokes Raman scattering) imaging technology, coherence between two molecular energy levels is firstly established by two beams of light (pumping light and Stokes light), and then signal light (anti-Stokes light) with characteristic spectral characteristics is excited by a third beam of light (detection light).
One of the solutions to the above problem is to use a time-resolved broad-spectrum CARS imaging technique. The technology uses a wide-spectrum femtosecond laser as pumping light, uses a narrow-spectrum wide picosecond laser with a certain time delay and frequency difference relative to the pumping light as detection light, and combines a frequency-resolved detection technology and a scanning imaging technology to realize rapid coherent Raman spectrum imaging without a non-resonance background. In the technology, because the ultra-wide spectrum short pulse laser is adopted, the coherence among a large number of molecular oscillation energy levels can be simultaneously established in a single pulse, so that the ultra-wide spectrum short pulse laser can be simultaneously used as pumping light and Stokes light of various molecules, the characteristic energy levels of various molecules can be excited without frequency scanning, the acquired data volume is greatly improved, and the ultra-wide spectrum imaging is realized; meanwhile, by adopting narrow-spectrum broad pulse laser, the frequency peak of Raman scattering light can be higher and narrower, and the non-resonance background spectrum is flatter, so that the non-resonance background is suppressed; furthermore, since the generation of the CARS signal is slower than the generation of the off-resonant background, the off-resonant background can be further eliminated using a probe laser pulse delayed from the pump laser pulse.
However, the conventional time-resolved broad-spectrum CARS device has high requirements on a light source, generally needs a complex Chirped Pulse Amplifier (CPA) and an Optical Parametric Amplifier (OPA), and the light source generally has a low repetition frequency, which greatly affects the imaging speed, and has a complex structure, a very high cost and is not easy to maintain.
Disclosure of Invention
Aiming at the defects of the prior art, the invention aims to provide a time-resolved broad-spectrum CARS spectral imaging device based on high-repetition-frequency femtosecond laser, and aims to solve the problems that the traditional CARS spectral imaging technology is difficult to realize single detection of various molecules and effectively inhibit the non-resonant background while keeping the system simple in structure and low in cost.
In order to achieve the aim, the invention provides a time-resolved broad spectrum CARS spectral imaging device based on high repetition frequency femtosecond laser, which comprises a high repetition frequency femtosecond laser, a double-pulse laser generation module and a spectral imaging platform;
the high repetition frequency femtosecond laser is used for generating fundamental frequency femtosecond laser with high repetition frequency, and the input double-pulse laser generating module is used for generating fundamental frequency light with high repetition frequency and inputting the fundamental frequency light into the double-pulse laser generating module;
the double-pulse laser generation module is used for generating coaxially transmitted fundamental frequency light and frequency shift light which are suitable for the time-resolved CARS imaging technology, and the fundamental frequency light and the frequency shift light are respectively used for pumping and detection;
the spectral imaging platform is used for realizing scanning type Raman spectral imaging on a sample to be detected.
Further, the spectral width of the fundamental frequency light is larger than that of the delay shift frequency light.
Further, the spectral imaging platform comprises: the first reflector and the second reflector are used for guiding the laser in a preset optimal position and angle; a first microscope objective for focusing the laser light reflected by the second mirror on the sample; the sample platform is used for fixing a sample and can realize three-dimensional scanning; the second microscope objective is used for collecting CARS signal light passing through the sample; the filter is used for filtering the fundamental frequency light and the frequency shift light and only allowing the CARS signal light to pass through; and the spectrometer is used for imaging the spectral information of the CARS signal light.
Further, the double-pulse laser generation module is a double-pulse laser generation module based on a space structure or a double-pulse laser generation module based on an optical fiber structure.
Preferably, when the double-pulse laser generation module is a spatial structure-based double-pulse laser generation module, the method includes: a focusing lens for focusing the fundamental frequency light to provide a sufficient high brightness for the frequency doubling process; the nonlinear element is used for converting part of the fundamental frequency light into frequency shift light which has a spectral width smaller than that of the fundamental frequency light and has a frequency difference with the fundamental frequency light; the collimating lens is used for collimating the transmitted divergent fundamental frequency light and the frequency shift light into parallel beams; the third reflector and the fourth reflector are used for collimating and folding the light path, so that the device is more compact in structure; the first dispersion compensation prism is used for spatially separating the fundamental frequency light and the frequency shift light, and after the fundamental frequency light is subjected to dispersion compensation, the two beams of light are combined through the fourth dispersion compensation prism; a narrow band filter for reducing a spectral width of the frequency-shifted light passing through the first dispersion compensating prism; the fifth reflector, the sixth reflector, the seventh reflector and the eighth reflector are connected in sequence and used for changing the time delay of the frequency-shifted light passing through the narrow-band filter relative to the fundamental frequency light, wherein the sixth reflector and the seventh reflector are controlled to be displaced by the displacement platform; and the half-wave plate is used for changing the polarization direction of the frequency-shifted light.
Preferably, when the double-pulse laser generation module is a fiber structure-based double-pulse laser generation module, the method comprises the following steps: the optical fiber beam splitter is used for splitting the fundamental frequency light into two paths; a first optical fiber amplifier and a second optical fiber amplifier for further amplifying the fundamental frequency light to provide sufficiently high optical pulse energy for subsequent nonlinear broadening; a first dispersion compensating fiber and a second dispersion compensating fiber for compensating dispersion to provide a sufficiently high peak power for subsequent nonlinear broadening; a first nonlinear optical fiber and a second nonlinear optical fiber for broadening the spectrum of the fundamental frequency light; the third optical fiber amplifier is used for amplifying a frequency spectrum component with frequency difference with the fundamental frequency light in the broadened spectrum of the fundamental frequency light to serve as frequency shift light; the optical fiber narrow-band filter is used for filtering other frequency spectrum components except the frequency shift light and further reducing the spectral width of the frequency shift light; a fourth optical fiber amplifier for further amplifying the frequency-shifted light; the ninth reflector, the tenth reflector, the eleventh reflector and the tenth reflector are connected in sequence and used for changing the time delay of the frequency shift light relative to the fundamental frequency light, and the displacement of the tenth reflector and the eleventh reflector is controlled by the displacement platform; the pulse compressor is used for compensating the chirp of the base frequency light after the spectrum broadening and compressing the pulse width; a fourteenth reflecting mirror for collimating the fundamental frequency light outputted from the fifth dispersion compensating prism, the sixth dispersion compensating prism and the thirteenth reflecting mirror into horizontal light and transmitting the laser light to a next element; and the dichroic mirror is used for combining the fundamental frequency light and the frequency shift light into one beam.
Preferably, the nonlinear element is a frequency doubling crystal or a photonic crystal fiber for generating resonant dispersion waves.
Furthermore, the sixth reflector and the seventh reflector change the time delay of the frequency-shifted light relative to the fundamental frequency light after passing through the narrow-band filter through front-back translation; the tenth reflector and the eleventh reflector change the time delay of the frequency-shifted light relative to the fundamental frequency light after passing through the optical fiber amplifier through back-and-forth translation.
Furthermore, the spectral imaging platform can be controlled by a PC (personal computer), and full-automatic synchronous mechanical scanning, data acquisition and data processing can be realized.
Furthermore, the spectrometer can be an imaging spectrometer, a dispersion Fourier transform spectrometer with higher spectrum acquisition speed, or a photomultiplier tube of a front filter for single-wavelength spectrum imaging.
Further, the pulse compressor may be a fifth dispersion compensation prism, a sixth dispersion compensation prism, and a thirteenth mirror connected in sequence, and may also be a chirped mirror, a grating compressor, or a fourier optical pulse shaper.
Through the technical scheme, compared with the prior art, the invention has the following beneficial effects:
(1) compared with the traditional CARS imaging technology, the invention adopts the femtosecond laser with wide spectrum as the pumping light and the Stokes light at the same time, and can establish the coherence among a plurality of energy levels at one time, thereby realizing the hyperspectral imaging of a plurality of molecules at the same time; the coherent excitation process and the detection light excitation process which are realized by the pump light and the Stokes light together are separated in time, and narrow spectrum light is adopted as the pump light, so that the non-resonant background is effectively inhibited, and the detection sensitivity is greatly improved;
(2) the time-resolved broad-spectrum CARS spectral imaging device based on the high-repetition-frequency femtosecond laser can realize the repetition frequency of hundreds of MHz or even GHz level without using CPA and OPA which are complex and high in price, so that the imaging speed is greatly improved;
(3) compared with the traditional optical imaging technology, the time-resolved broad-spectrum CARS spectral imaging device based on the high-repetition-frequency femtosecond laser has chemical specificity, and can excite CARS signal light with different wavelengths aiming at different molecules, so that specific molecules can be distinguished according to the spectral characteristics of the generated signal light, and the spatial distribution of the specific molecules can be obtained through a scanning imaging mode;
(4) compared with the incoherent Raman spectrum imaging technology, the time-resolved broad spectrum CARS spectrum imaging device based on the high-repetition-frequency femtosecond laser greatly improves the generation efficiency of signal light, thereby reducing the integration time required by signal light acquisition and further realizing higher imaging speed;
(5) the process of exciting the signal light by the high-repetition-frequency femtosecond laser-based time-resolved broad-spectrum CARS spectral imaging device is a multi-photon process and can only occur in a small region with extremely high intermediate energy of a focused light spot, so that the device has extremely high spatial resolution naturally, and if the device is used in a transparent medium, high-resolution three-dimensional imaging can even be realized through three-dimensional space scanning;
(6) compared with the fluorescence labeling imaging technology, the time-resolved broad spectrum CARS spectral imaging device based on the high-repetition-frequency femtosecond laser can realize label-free imaging, does not need to pretreat a sample, is simpler and more convenient to operate, and does not cause modification of the sample;
(7) compared with the infrared imaging technology, the invention generally adopts near infrared light with shorter wavelength to scan, such as a titanium gem femtosecond laser, an erbium-doped femtosecond fiber laser or an ytterbium-doped femtosecond fiber laser, the output wavelength of which is in a near infrared light wave band, on one hand, the light focusing spot with shorter wavelength is smaller under the same focusing condition, which can improve the spatial resolution of scanning imaging; on the other hand, water absorbs near infrared light more than mid-infrared light, and thus the present invention is more suitable for imaging moisture-rich biological samples than infrared imaging techniques.
Drawings
FIG. 1 is a schematic structural diagram of a high-repetition-frequency femtosecond laser-based time-resolved broad-spectrum CARS spectral imaging device provided by the invention;
fig. 2 is a schematic structural diagram of a double-pulse laser generating module according to an embodiment of the present invention;
fig. 3 is a schematic structural diagram of a double-pulse laser generating module according to a second embodiment of the present invention.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is described in further detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention. In addition, the technical features involved in the embodiments of the present invention described below may be combined with each other as long as they do not conflict with each other.
As shown in fig. 1, the invention provides a time-resolved broad spectrum CARS spectral imaging device based on a high repetition frequency femtosecond laser, which comprises a high repetition frequency femtosecond laser, a double-pulse laser generation module and a spectral imaging platform;
the high repetition frequency femtosecond laser is used for generating fundamental frequency femtosecond laser with high repetition frequency, inputting the fundamental frequency femtosecond laser into the double-pulse laser generation module and providing enough high light intensity and single-pulse energy for the double-pulse laser generation;
the double-pulse laser generation module is used for generating coaxially transmitted fundamental frequency light and frequency shift light which are suitable for the time-resolved CARS imaging technology, and the fundamental frequency light and the frequency shift light are respectively used for pumping and detection;
the spectral imaging platform is used for realizing scanning type Raman spectral imaging on a sample to be detected.
The high repetition frequency fundamental frequency femtosecond laser generated by the high repetition frequency femtosecond laser 1 is converted into a double pulse laser suitable for a time resolution wide spectrum CARS imaging technology after passing through a double pulse laser generating module 2, wherein the double pulse laser comprises a beam of wide spectrum fundamental frequency light (as pumping light of the CARS imaging technology) and a beam of narrow spectrum frequency shift light (as probe light of the CARS imaging technology) with proper frequency difference and time delay with the pumping light pulse, and the two beams of light are combined into a beam for coaxial transmission; the double-pulse laser is guided into a first microscope objective 5 of the spectral imaging platform through a first reflector 3 and a second reflector 4; the first microscope objective 5 tightly focuses incident light on a sample fixed on a sample platform 6, generated CARS signal light is collected by a second microscope objective 7, and after passing through a filter 8, fundamental frequency light and frequency shift light are filtered, and only the CARS signal light enters an imaging spectrometer consisting of a spectrometer 9 and a high-performance camera 10, so that the acquisition of signal light spectrum information is completed; and the mechanical scanning of the sample platform 6, the data acquisition of the imaging spectrometer and the data processing of the computer are synchronously controlled by software, so that a visual spectral imaging result can be obtained.
Fig. 2 is a schematic structural diagram of a double-pulse laser generating module based on a spatial structure according to a first embodiment of the present invention, in which a part of fundamental frequency light output by a high repetition frequency femtosecond laser 1 is converted into frequency shift light that is still coaxially transmitted with the fundamental frequency light after passing through a focusing lens 11, a nonlinear element 12, and a collimating lens 13, and the frequency shift light is guided to a first dispersion prism 16 through a third reflector 14 and a fourth reflector 15; the first dispersion prism 16 divides the original coaxially transmitted fundamental frequency light and frequency shift light into two paths, and the fundamental frequency light continues to pass through the second dispersion compensation prism 17, the third dispersion compensation prism 18 and the fourth dispersion compensation prism 19, so that dispersion compensation of the fundamental frequency light is completed, and ultra-short laser pulse output is realized; the frequency-shifted light sequentially passes through a narrow-band filter 20 for compressing spectrum width, a retarder for adjusting time delay and a half-wave plate 25 for adjusting polarization direction, wherein the retarder is composed of a fifth mirror 21-24, a sixth mirror 21-24, a seventh mirror and an eighth mirror 26; the fundamental light and the frequency-shifted light are combined again into one beam at the fourth dispersion compensating prism 19.
Fig. 3 is a schematic structural diagram of a double-pulse laser generating module based on a space structure and based on an optical fiber structure according to a second embodiment of the present invention, in which fundamental frequency light output by the high repetition frequency femtosecond laser 1 is transmitted to the optical fiber beam splitter 27 via an optical fiber and is divided into two paths; one path of light is amplified by a first optical fiber amplifier 28, the pulse width of the first dispersion compensation optical fiber 30 is compressed, the amplified light is input into a first nonlinear optical fiber 32 to be subjected to sufficient spectrum broadening, narrow-band frequency shift light used as CARS detection light is selected and amplified by a third optical fiber amplifier 34, an optical fiber narrow-band filter 35 and a fourth optical fiber amplifier 36, and finally the amplified light passes through a delayer which consists of a ninth reflector 37, a tenth reflector 37-40, an eleventh reflector 37-40 and a displacement platform 41 and is used for adjusting the time delay relative to the other path of laser pulse and is incident on a dichroic mirror 42; the other path of light is amplified by the second optical fiber amplifier 29, the pulse width of the second dispersion compensation fiber 31 is compressed, the other path of light is input into the second nonlinear optical fiber 33 for sufficient spectral broadening, the output light grazes the upper edge of the thirteenth reflecting mirror 43, enters a pulse compressor consisting of the fifth dispersion compensation prism 44, the sixth dispersion compensation prism 45 and the fourteenth reflecting mirror 46, returns in the same horizontal direction at a slightly lower angle, can be incident on the mirror surface of the thirteenth reflecting mirror 43 at this time, and is readjusted to be horizontal light by the thirteenth reflecting mirror 43 to be incident on the dichroic mirror 42; the narrow spectrum frequency shift light is transmitted through the dichroic mirror 42, the wide spectrum fundamental frequency light is reflected by the dichroic mirror 42, and the two beams of light are combined into one coaxial beam and enter the rear spectral imaging platform.
It will be understood by those skilled in the art that the foregoing is only a preferred embodiment of the present invention, and is not intended to limit the invention, and that any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the scope of the present invention.
Claims (8)
1. A time-resolved broad-spectrum CARS spectral imaging device based on high-repetition-frequency femtosecond laser is characterized by comprising a high-repetition-frequency femtosecond laser, a double-pulse laser generation module and a spectral imaging platform;
the high repetition frequency femtosecond laser is used for generating fundamental frequency light with high repetition frequency and inputting the fundamental frequency light into the double-pulse laser generation module;
the double-pulse laser generation module is used for generating coaxially transmitted fundamental frequency light and frequency shift light which are suitable for the time-resolved CARS imaging technology, and the fundamental frequency light and the frequency shift light are respectively used for pumping and detection; the system is divided into a double-pulse laser generating module based on a space structure and a double-pulse laser generating module based on an optical fiber structure;
the double-pulse laser generation module based on the space structure comprises: a focusing lens for focusing the fundamental frequency light; the nonlinear element is used for converting part of the fundamental frequency light into frequency shift light which has a spectral width smaller than that of the fundamental frequency light and has a frequency difference with the fundamental frequency light; the collimating lens is used for collimating the transmitted divergent fundamental frequency light and the frequency shift light into parallel beams; the third reflector and the fourth reflector are used for collimating the light path and folding the light path; the first dispersion compensation prism is used for spatially separating fundamental frequency light and frequency shift light, and after the fundamental frequency light is subjected to dispersion compensation, the two beams of light are combined through the fourth dispersion compensation prism; a narrow band filter for reducing a spectral width of the frequency-shifted light passing through the first dispersion compensating prism; the fifth reflector, the sixth reflector, the seventh reflector and the eighth reflector are connected in sequence and used for changing the time delay of the frequency-shifted light passing through the narrow-band filter relative to the fundamental frequency light; the half-wave plate is used for changing the polarization direction of the frequency-shifted light;
the double-pulse laser generation module based on the optical fiber structure comprises: the optical fiber beam splitter is used for splitting the fundamental frequency light into two paths; a first optical fiber amplifier and a second optical fiber amplifier for further amplifying the fundamental frequency light; a first dispersion compensating fiber and a second dispersion compensating fiber for compensating dispersion; a first nonlinear optical fiber and a second nonlinear optical fiber for broadening the spectrum of the fundamental frequency light; the third optical fiber amplifier is used for amplifying a frequency spectrum component with frequency difference with the fundamental frequency light in the broadened spectrum of the fundamental frequency light to serve as frequency shift light; the optical fiber narrow-band filter is used for filtering other frequency spectrum components except the frequency shift light and further reducing the spectral width of the frequency shift light; a fourth optical fiber amplifier for further amplifying the frequency-shifted light; the ninth reflector, the tenth reflector, the eleventh reflector and the tenth reflector are connected in sequence and used for changing the time delay of the frequency shift light relative to the fundamental frequency light; the pulse compressor is used for compensating the chirp of the base frequency light after the spectrum broadening and compressing the pulse width; a fourteenth reflecting mirror for collimating the fundamental frequency light outputted from the pulse compressor into horizontal light and transmitting the laser light to a next element; the dichroic mirror is used for combining the fundamental frequency light and the frequency shift light into one beam;
the spectral imaging platform is used for realizing scanning type Raman spectral imaging on a sample to be detected.
2. The apparatus of claim 1, wherein the spectral width of the fundamental light is greater than the spectral width of the frequency-shifted light.
3. The apparatus of claim 2, wherein the spectral imaging stage comprises: the laser device comprises a first reflector and a second reflector, wherein the first reflector and the second reflector are used for guiding laser in at preset positions and angles; the first microscope objective is used for focusing the laser reflected by the second reflector on the sample; the sample platform is used for fixing a sample and can realize three-dimensional scanning; the second microscope objective is used for collecting CARS signal light passing through the sample; the filter is used for filtering the fundamental frequency light and the frequency shift light and only allowing the CARS signal light to pass through; and the spectrometer is used for imaging the spectral information of the CARS signal light.
4. The apparatus of claim 1, wherein the nonlinear element is a frequency doubling crystal or a photonic crystal fiber for generating resonant dispersion waves.
5. The apparatus of claim 1, wherein the sixth mirror and the seventh mirror change the time delay of the frequency-shifted light relative to the fundamental light after passing through the narrow band filter by translation.
6. The apparatus of claim 1, wherein the tenth mirror and the eleventh mirror change the time delay of the frequency-shifted light relative to the fundamental light after passing through the fiber amplifier by translation.
7. The apparatus of claim 1, wherein the spectrometer is an imaging spectrometer, a dispersive fourier transform spectrometer, or a pre-filter photomultiplier tube.
8. The apparatus of claim 1, wherein the pulse compressor is a fifth dispersion compensating prism, a sixth dispersion compensating prism, and a thirteenth mirror connected in sequence; or any of a chirped mirror, a grating compressor, a fourier optical pulse shaper.
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