CN110579462B - A time-resolved broad-spectrum CARS spectral imaging device based on high repetition frequency femtosecond laser - Google Patents

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

A time-resolved broad-spectrum CARS spectral imaging device based on high repetition frequency femtosecond laser

technical field

The invention belongs to the technical field of optical imaging, and more particularly, relates to a time-resolved broad-spectrum CARS spectral imaging device based on a high repetition frequency femtosecond laser.

Background technique

Optical imaging technology has a wide range of applications in materials science, biomedicine and other fields. At present, there are still many problems in traditional optical imaging technology. For example, general optical imaging lacks chemical specificity and cannot image the spatial distribution of a specific molecule in a sample. This lack of chemical information will limit the application of imaging technology to a large extent; although fluorescent labeling imaging has chemical specificity However, it requires the labeling of specific molecules, that is, complex pretreatment of the sample is required, which is not only time-consuming and labor-intensive, but also may change the characteristics of the sample. Moreover, there is currently a lack of labeling methods for most molecules; Raman spectroscopy imaging Label-free molecular imaging can be achieved, but limited by the extremely low efficiency of Raman scattering, a sufficiently long integration time is required to acquire a signal with a sufficiently high signal-to-noise ratio, which greatly limits the imaging speed; CARS (Coherent Anti-Stokes Raman Scattering, Coherent anti-Stokes Raman scattering) imaging technology uses two beams of light (pump light and Stokes light) to first establish the coherence between the energy levels of the two molecules, and then excited by the third beam (probe light). Signal light with characteristic spectral characteristics (anti-Stokes light), this method can greatly improve the efficiency of Raman scattering, but will produce a flat and broad non-resonant background spectrum, and sometimes even overwhelm the CARS spectral peaks, affecting imaging In addition, traditional CARS imaging devices generally use two narrow-spectrum picosecond lasers with different wavelengths. One beam acts as pump light and probe light at the same time, and the other beam acts as Stokes light, which can only be excited each time. A molecule that produces anti-Stokes light of a single wavelength, making it impossible to see the distribution of multiple molecules at the same time.

One of the ways to solve the above problems is to use time-resolved broad-spectrum CARS imaging technology. This technology uses a broad-spectrum femtosecond laser as the pump light, and a narrow-spectrum picosecond with a certain delay and frequency difference relative to the pump light as the probe light. Combined with the frequency-resolved detection technology and scanning imaging technology, it can achieve fast , Coherent Raman spectroscopy imaging without non-resonant background. In this technology, because the ultra-broad-spectrum short-pulse laser is used, the coherence between a large number of molecular vibrational and rotational energy levels can be established simultaneously in a single pulse, so it can act as the pump light and the laser light for a variety of molecules at the same time. Tox light can excite the characteristic energy levels of a variety of molecules without frequency scanning, which greatly increases the amount of data collected, enabling so-called "hyperspectral imaging"; at the same time, a narrow-spectrum pulsed laser , the frequency peak of Raman scattered light can be made higher and narrower, and the off-resonance background spectrum is flatter, thereby suppressing the off-resonance background; in addition, since the generation of the CARS signal is slower than that of the off-resonance background, the use of a delay of The probe laser pulse of the pump laser pulse can further eliminate the non-resonant background.

However, the traditional time-resolved broad-spectrum CARS device has high requirements on the light source, and generally requires the use of complex chirped pulse amplifier (CPA) and optical parametric amplifier (OPA). This light source generally has a low repetition frequency, which will greatly affect the imaging speed. , and the structure is complex, the cost is very high, and it is not easy to maintain.

SUMMARY OF THE INVENTION

In view of the defects of the prior art, the purpose of the present invention is to provide a time-resolved broad-spectrum CARS spectral imaging device based on a high repetition frequency femtosecond laser, which aims to solve the problem that the traditional CARS spectral imaging technology is difficult to maintain a simple system structure and a low cost. At the same time, it can detect multiple molecules at a time, and effectively suppress the problem of non-resonant background.

In order to achieve the above object, the present invention provides a time-resolved broad-spectrum CARS spectral imaging device based on a high repetition frequency femtosecond laser, including 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 to generate the fundamental frequency femtosecond laser with high repetition frequency, and input into the double pulse laser generation module, which is used to generate the fundamental frequency light of high repetition frequency and input into the double pulse laser generation module;

The dual-pulse laser generation module is used to generate coaxial transmission of fundamental frequency light and frequency-shifted light suitable for time-resolved CARS imaging technology. The fundamental frequency light and the frequency-shifted light are used for pumping and detection respectively;

The spectral imaging platform is used to realize scanning Raman spectral imaging of the sample to be tested.

Further, the spectral width of the fundamental frequency light is larger than that of the delayed frequency-shifted light.

Further, the spectral imaging platform includes: a first reflecting mirror and a second reflecting mirror for guiding the laser light at a preset optimal position and angle; a first microscope objective lens for focusing the laser light reflected by the second reflecting mirror On the sample; a sample platform for fixing the sample and enabling 3D scanning; a second microscope objective for collecting the CARS signal light passing through the sample; a filter for filtering the fundamental frequency light and the frequency-shifted light, allowing only The CARS signal light passes through; a spectrometer for imaging the spectral information of the CARS signal light.

Further, the double-pulse laser generating module is a space-structure-based double-pulse laser generating module or an optical fiber structure-based double-pulse laser generating module.

Preferably, when the double-pulse laser generating module is a double-pulse laser generating module based on a spatial structure, it includes: a focusing lens for focusing the fundamental frequency light so as to provide sufficient high brightness for the frequency doubling process; a nonlinear element for Part of the fundamental frequency light is converted into frequency-shifted light whose spectral width is smaller than that of the fundamental-frequency light and has a frequency difference with the fundamental-frequency light; the collimating lens is used to collimate the transmitted divergent fundamental-frequency light and the frequency-shifted light into parallel beams; The third reflection mirror and the fourth reflection mirror are used for collimating the optical path and folding the optical path, so that the structure of the device is more compact; the first dispersion compensation prism, the second dispersion compensation prism, the third dispersion compensation prism and the fourth dispersion compensation prism are connected in sequence. Dispersion compensation prism, the first dispersion compensation prism is used to spatially separate the fundamental frequency light from the frequency-shifted light. After the fundamental frequency light is dispersion compensated, the two beams are combined by the fourth dispersion compensation prism; the narrow-band filter is used to reduce the The spectral width of the frequency-shifted light passing through the first dispersion compensation prism; the fifth, sixth, seventh and eighth mirrors connected in sequence are used to change the frequency-shifted light after passing through the narrowband filter Relative to the time delay of the fundamental frequency light, the displacement of the sixth mirror and the seventh mirror is controlled by the displacement platform; the half-wave plate is used to change the polarization direction of the frequency-shifted light.

Preferably, when the double-pulse laser generating module is a double-pulse laser generating module based on an optical fiber structure, it includes: a fiber beam splitter, which is used to divide the fundamental frequency light into two paths; a first fiber amplifier and a second fiber amplifier, using to further amplify the fundamental frequency light to provide sufficiently high optical pulse energy for subsequent nonlinear broadening; the first dispersion compensating fiber and the second dispersion compensating fiber are used to compensate for dispersion to provide sufficiently high peak power for subsequent nonlinear broadening ; The first nonlinear optical fiber and the second nonlinear optical fiber are used to broaden the spectrum of the fundamental frequency light; the third optical fiber amplifier is used to amplify the spectral components that have a frequency difference with the fundamental frequency light in the broadened spectrum of the fundamental frequency light, as a shift frequency light; fiber narrow-band filter, used to filter out other spectral components other than frequency-shifted light, and further reduce the spectral width of frequency-shifted light; fourth fiber amplifier, used to further amplify frequency-shifted light; The nine reflection mirrors, the tenth reflection mirror, the eleventh reflection mirror and the twelfth reflection mirror are used to change the time delay of the frequency-shifted light relative to the fundamental frequency light, wherein the tenth reflection mirror and the eleventh reflection mirror are determined by the displacement platform Control displacement; pulse compressor, used for compensating the chirp of the fundamental frequency light after spectrum broadening, and compressing the pulse width; the fourteenth reflection mirror, used for combining the fifth dispersion compensation prism, the sixth dispersion compensation prism and the thirteenth dispersion compensation prism The fundamental frequency light output by the mirror is collimated into horizontal light, and the laser is transmitted to the next element; the dichroic mirror is used to combine the fundamental frequency light and the frequency-shifted light into one beam.

Preferably, the nonlinear element is a frequency-doubling crystal or a photonic crystal fiber for generating resonant dispersion waves.

Further, the sixth reflection mirror and the seventh reflection mirror change the time delay of the frequency-shifted light after passing through the narrowband filter relative to the fundamental frequency light by shifting back and forth; The time delay of the frequency-shifted light after the amplifier relative to the fundamental frequency light.

Further, the spectral imaging platform can be controlled by a PC, enabling fully automatic synchronous mechanical scanning, data acquisition and data processing.

Further, the spectrometer can be an imaging spectrometer, a dispersive Fourier transform spectrometer with a faster spectral collection speed, or a photomultiplier tube with a pre-filter for single-wavelength spectral imaging.

Further, the pulse compressor may be the fifth dispersion compensating prism, the sixth dispersion compensating prism and the thirteenth reflection mirror connected in sequence, and may also be a chirped mirror, a grating compressor or a Fourier light pulse shaper.

Through the above technical solutions conceived by the present invention, compared with the prior art, the following beneficial effects can be achieved:

(1) Compared with the traditional CARS imaging technology, the present invention uses a broad-spectrum femtosecond laser as the pump light and the Stokes light at the same time, and can establish the coherence between multiple energy levels at one time, so that the simultaneous imaging can be realized. Hyperspectral imaging of a variety of molecules; the coherent excitation process and the probe light excitation process jointly realized by the pump light and Stokes light are separated in time, and the narrow-spectrum light is used as the pump light, which effectively suppresses the The resonance background greatly improves the detection sensitivity;

(2) The time-resolved broad-spectrum CARS spectral imaging device based on the high repetition frequency femtosecond laser provided by the present invention can achieve a repetition frequency of hundreds of MHz or even GHz without using complex and expensive CPA and OPA, thereby greatly reducing the need for Improved imaging speed;

(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 provided by the present invention has chemical specificity, and can excite the CARS signal light of different wavelengths for different molecules, thereby Specific molecules can be identified according to the spectral characteristics of the generated signal light, and then the spatial distribution of specific molecules can be obtained by scanning and imaging;

(4) Compared with the incoherent Raman spectral imaging technology, the time-resolved broad-spectrum CARS spectral imaging device based on the high repetition frequency femtosecond laser provided by the present invention greatly improves the efficiency of signal light generation, thereby reducing the time required for collecting signal light. required integration time, which in turn enables faster imaging;

(5) The process of exciting the signal light of the time-resolved broad-spectrum CARS spectral imaging device based on the high repetition frequency femtosecond laser provided by the present invention is a multi-photon process, which occurs only in a small area with extremely high energy in the middle of the focused spot, so the natural It has extremely high spatial resolution. If it is used in transparent media, it can even achieve high-resolution 3D imaging by scanning in 3D space;

(6) Compared with the fluorescent labeling imaging technology, the time-resolved broad-spectrum CARS spectral imaging device based on the high repetition frequency femtosecond laser provided by the present invention can realize label-free imaging, does not need to preprocess the sample, and the operation is more convenient, And will not cause modification of the sample;

(7) Compared with infrared imaging technology, the present invention generally uses near-infrared light with shorter wavelength for scanning, such as Ti:sapphire femtosecond laser, erbium-doped femtosecond fiber laser or ytterbium-doped femtosecond fiber laser, whose output wavelength is at In the near-infrared light band, on the one hand, under the same focusing conditions, light with a shorter wavelength has a smaller focused spot, which can improve the spatial resolution of scanning imaging; on the other hand, water absorbs near-infrared light more than mid-infrared light. Therefore, the present invention is more suitable for imaging water-rich biological samples than infrared imaging technology.

Description of drawings

1 is a schematic structural diagram of a time-resolved broad-spectrum CARS spectral imaging device based on a high repetition frequency femtosecond laser provided by the present invention;

2 is a schematic structural diagram of a dual-pulse laser generating module provided in Embodiment 1 of the present invention;

FIG. 3 is a schematic structural diagram of a double-pulse laser generating module provided in Embodiment 2 of the present invention.

Detailed ways

In order to make the objectives, technical solutions and advantages of the present invention clearer, the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention, but not to limit the present invention. In addition, the technical features involved in the various embodiments of the present invention described below can be combined with each other as long as they do not conflict with each other.

As shown in FIG. 1 , the present invention provides a time-resolved broad-spectrum CARS spectral imaging device based on a high repetition frequency femtosecond laser, including a high repetition frequency femtosecond laser, a double-pulse laser generation module and a spectral imaging platform;

High repetition frequency femtosecond laser, used to generate fundamental frequency femtosecond laser with high repetition rate, input into the double-pulse laser generation module to provide sufficiently high light intensity and single-pulse energy for double-pulse laser generation;

The dual-pulse laser generation module is used to generate coaxial transmission of fundamental frequency light and frequency-shifted light suitable for time-resolved CARS imaging technology. The fundamental frequency light and the frequency-shifted light are used for pumping and detection respectively;

The spectral imaging platform is used to realize scanning Raman spectral imaging of the sample to be tested.

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 the time-resolved broad-spectrum CARS imaging technology after passing through the double pulse laser generation module 2, which includes a A beam of broad-spectrum fundamental frequency light (as the pump light of CARS imaging technology) and a beam of narrow-spectrum frequency-shifted light (as the probe light of CARS imaging technology) with appropriate frequency difference and time delay with the pump light pulse, two beams of light be combined into a coaxial transmission; the double-pulse laser is introduced into the first microscope objective lens 5 of the spectral imaging platform via the first reflector 3 and the second reflector 4; the first microscope objective lens 5 tightly focuses the incident light On the sample fixed on the sample platform 6, the generated CARS signal light is collected by the second microscope objective lens 7, and after passing through the filter 8, the fundamental frequency light and the frequency-shifted light are filtered out, and only the CARS signal light is allowed to enter the spectrometer 9. In the imaging spectrometer composed of the high-performance camera 10, the acquisition of the spectral information of the signal light is completed; the mechanical scanning of the sample platform 6, the data acquisition of the imaging spectrometer and the data processing of the computer can be controlled by the software to obtain the visual spectral imaging. result.

2 is a schematic structural diagram of a double-pulse laser generation module based on a spatial structure provided in Embodiment 1 of the present invention. The fundamental frequency light output by the high repetition frequency femtosecond laser 1 passes through a focusing lens 11 , a nonlinear element 12 and a collimating lens 13 After that, part of it is converted into frequency-shifted light that still transmits coaxially with the fundamental frequency light, and is guided to the first dispersion prism 16 via the third reflection mirror 14 and the fourth reflection mirror 15; the first dispersion prism 16 transmits the original coaxial transmission The fundamental frequency light and the frequency-shifted light are divided 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 as to complete the dispersion compensation of the fundamental frequency light, and then Realize ultra-short laser pulse output; the frequency-shifted light sequentially passes through the narrow-band filter 20 for compressing the spectral width, the The extended retarder and the half-wave plate 25 for adjusting the polarization direction; the fundamental frequency light and the shifted frequency light are combined into one beam again at the fourth dispersion compensation prism 19 .

3 is a schematic structural diagram of a double-pulse laser generation module based on an optical fiber structure based on a spatial structure provided in Embodiment 2 of the present invention, and the fundamental frequency light output by the high repetition frequency femtosecond laser 1 is transmitted to the optical fiber beam splitter 27 via the optical fiber, It is divided into two paths; one path of light is amplified by the first fiber amplifier 28, and after the pulse width is compressed by the first dispersion compensation fiber 30, it is input into the first nonlinear fiber 32 for sufficient spectral broadening, and is amplified by the third fiber amplifier 34. , the fiber narrowband filter 35 and the fourth fiber amplifier 36 select and amplify the narrowband frequency-shifted light used as the CARS probe light, and finally pass through the ninth, tenth, eleventh, and twelfth mirrors 37-40 and displacement The retarder composed of the platform 41 and used to adjust the time delay relative to another laser pulse is incident on the dichroic mirror 42; the other light is amplified by the second fiber amplifier 29, and the second dispersion compensation fiber 31 compresses the pulse width after the , input into the second nonlinear fiber 33 for sufficient spectral broadening, after the output light grazing the upper edge of the thirteenth reflection mirror 43, enters the fifth dispersion compensation prism 44, the sixth dispersion compensation prism 45 and the tenth dispersion compensation prism 43. In the pulse compressor composed of four mirrors 46, and returning to the same horizontal direction at a slightly lower angle, it can be incident on the mirror surface of the thirteenth mirror 43, and the thirteenth mirror 43 readjusts The horizontal light is incident on the dichroic mirror 42; the narrow-spectrum frequency-shifted light is transmitted through the dichroic mirror 42, the broad-spectrum fundamental frequency light is reflected by the dichroic mirror 42, and the two beams of light are combined into a coaxial beam of light and incident into the rear spectral imaging platform.

Those skilled in the art can easily understand that the above are only preferred embodiments of the present invention, and are not intended to limit the present invention. Any modifications, equivalent replacements and improvements made within the spirit and principles of the present invention, etc., All should be included within the protection 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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