Coherent anti-stokes Raman microscope system, driving method thereof and microscopic device

Abstract

The invention provides a coherent anti-Stokes Raman microscope system, a driving method thereof and a microscopic device. The coherent anti-Stokes Raman microscope system comprises a femtosecond laser front-end module, a dual-wavelength module, a pulse broadening amplification module, a time delay module, a spectrum broadening filtering module, a double frequency generation module, a dispersion compensation module and a microscope module. According to the coherent anti-Stokes Raman microscope system driven by self-phase modulation spectrum selection, a self-phase modulation spectrum selection (SESS) method is adopted to convert the pulse wavelength of 1550nm to 1600nm, a second harmonic generation process is realized through a frequency doubling crystal, the pulse wavelength of 1600nm is converted to 800nm, coherent anti-Stokes Raman microscopic imaging is realized, and the coherent anti-Stokes Raman microscope system driven by self-phase modulation spectrum selection has the advantages that the coherent anti-Stokes Raman microscope system is simple in structure and convenient to operate. Therefore, selective imaging of various biological tissue components is realized. Compared with other wavelength conversion methods, the device is simple, the conversion efficiency is high, the wavelength is tunable, and the expansibility is high.

Description

Technical Field

[0001] The invention belongs to the technical field of ultrafast lasers, and in particular relates to a coherent anti-Stokes Raman microscope system, a driving method thereof and a microscope device. Background Art

[0002] Coherent anti-Stokes Raman scattering (CARS) is a label-free optical microscopy imaging method based on the vibration of molecular chemical bonds. It is widely used in biological tissue imaging and pharmacokinetics due to its characteristics of label-free, strong specificity and low photodamage.

[0003] CARS imaging requires pump light and Stokes light. The two beams of light overlap in space and time and are focused onto the sample through a microscope. When the frequency difference between the two is consistent with the molecular vibration frequency, an anti-Stokes light signal is generated.

[0004] In order to achieve selective imaging and increase the signal-to-noise ratio, the driving light source should be a picosecond (ps) laser with tunable wavelength. Currently, most of the existing driving light sources are realized through soliton self-frequency shift, electrical modulation, four-wave mixing and other methods, but the current light source realization technologies have their own defects.

[0005] Defects of the prior art:

[0006] 1. Wavelength conversion technologies based on four-wave mixing, such as optical parametric oscillator / optical parametric amplifier, have very low conversion efficiency and are very sensitive to environmental fluctuations.

[0007] 2. The output pulse energy of wavelength conversion technologies based on soliton self-frequency shift and supercontinuum is limited. Summary of the invention

[0008] Therefore, the purpose of the present invention is to overcome the defects in the prior art and provide a coherent anti-Stokes Raman microscope system, a driving method thereof and a microscope device. The CARS microscope system of the present invention is based on the self-phase modulation spectral selection (SESS) of the dual-wavelength system and the second harmonic ultrafast fiber laser light source to drive the CARS system. Compared with the wavelength conversion method in the prior art, the nonlinear wavelength conversion technology SESS used in this device can output pulses with high conversion efficiency and fully compressible pulse width.

[0009] Before describing the present invention, the terms used in this article are defined as follows:

[0010] The term "CARS" refers to: Coherent Anti-Stokes Raman Microscopy.

[0011] The term "LMA-8" refers to: Large Mode Area Photonic Crystal Fiber-8.

[0012] The term "ESM-12" means: Single Mode Photonic Crystal Fiber-12.

[0013] The term "SESS" means: Self-Phase Modulation Spectral Selection.

[0014] The term "P-polarization" refers to the phenomenon that when light strikes the surface of an optical component (such as a beamsplitter) at a non-perpendicular angle, both the reflection and transmission characteristics are polarization dependent. In this case, the coordinate system used is defined by the plane containing the input and reflected beams. If the polarization vector of the light lies in this plane, it is called P-polarization.

[0015] The term "LEAF fiber" refers to: Large Effective-Area Fiber, a single-mode dispersion-shifted fiber that has a larger "effective area" than standard dispersion-shifted fiber. Its effective area is ≥72um. 2 , so it has a larger power handling capacity and is suitable for use with high output power erbium-doped fiber amplifiers.

[0016] The term "BBO" means: barium metaborate.

[0017] The term "PPLN" means: Periodically Poled Lithium Niobate.

[0018] The term "Yb401 single-mode fiber amplifier" means: a Yb-doped single-mode fiber amplifier.

[0019] The term "Yb12 / 125 multimode fiber amplifier" refers to: a Yb-doped multimode fiber amplifier.

[0020] The term "Yb30 / 250 Multimode Fiber Amplifier" refers to: Another Yb-doped multimode fiber amplifier.

[0021] The term "Er80 single-mode fiber amplifier" refers to: an Er-doped single-mode fiber amplifier.

[0022] The term "Er40 single-mode fiber amplifier" refers to: another type of Er-doped single-mode fiber amplifier.

[0023] The term "Yb / Er30 / 250 multimode fiber amplifier" refers to: a Yb / Er co-doped multimode fiber amplifier.

[0024] To achieve the above object, a first aspect of the present invention provides a coherent anti-Stokes Raman microscope system, the coherent anti-Stokes Raman microscope system comprising:

[0025] Femtosecond laser front-end module for outputting ultrashort pulse trains,

[0026] A dual-wavelength module for broadening the spectrum of the ultrashort pulse sequence,

[0027] A pulse stretching and amplification module is used to introduce dispersion into the stretched pulse to stretch the pulse width and then amplify the power.

[0028] Time delay module used to compensate for the optical path difference of two pulses.

[0029] A spectrum broadening filter module is used to filter the spectrum after broadening the pulse width output by the pulse broadening and amplification module,

[0030] A frequency doubling generation module for doubling the frequency of the pulse output from the spectrum broadening and filtering module,

[0031] A dispersion compensation module for compensating the dispersion time-domain broadening of two pulsed optical beams, and

[0032] Microscope module;

[0033] Furthermore, the output end of the femtosecond laser front-end module is connected to the input end of the dual-wavelength module, the output end of the dual-wavelength module is connected to the input end of the pulse stretching and amplification module, the output end of the pulse stretching and amplification module is connected to the input end of the time delay module and the spectrum stretching and filtering module, the output end of the spectrum stretching and filtering module is connected to the input end of the double frequency generation module, the output ends of the double frequency generation module and the time delay module are connected to the input end of the dispersion compensation module, and the output end of the dispersion compensation module is connected to the input end of the microscope module;

[0034] Preferably, the spectrum broadening of the spectrum broadening filter module is achieved through a self-phase modulation effect, and / or

[0035] Preferably, the multiphoton microscope module is driven by pulses output by the dispersion compensation module to image the sample.

[0036] According to the coherent anti-Stokes Raman microscope system of the first aspect of the present invention, the femtosecond laser front-end module comprises an oscillator; wherein,

[0037] The repetition frequency of the femtosecond pulse sequence output by the femtosecond laser front-end module is 35-40 MHz, and most preferably 37 MHz;

[0038] The central wavelength range of the ultrashort pulse sequence emitted by the oscillator in the femtosecond laser front-end module is 1.50 μm to 1.66 μm, preferably 1.54 μm to 1.57 μm, and most preferably 1.55 μm; and / or

[0039] The oscillator is a fiber laser oscillator or a solid-state laser oscillator, preferably an erbium-doped fiber laser oscillator, more preferably a mode-locked erbium-doped fiber laser oscillator;

[0040] Preferably, the mode locking method is selected from one or more of the following: semiconductor saturable absorber mirror, nonlinear polarization rotation, nonlinear optical loop mirror, and most preferably semiconductor saturable absorber mirror.

[0041] The coherent anti-Stokes Raman microscope system according to the first aspect of the present invention, wherein:

[0042] The dual-wavelength module includes in sequence: a dual-wavelength optical fiber isolator, a dual-wavelength pump laser source, a dual-wavelength wavelength division multiplexer, a dual-wavelength optical fiber amplifier and a high nonlinear optical fiber; and / or

[0043] The time delay module includes an optical path compensation device; wherein the optical path compensation device includes: an optical path compensation reflector and an optical path compensation translation stage;

[0044] Preferably, the optical path compensation reflector is mounted on the optical path compensation translation stage; and / or

[0045] Preferably, the number of the optical path compensation reflectors is 2 to 10, more preferably 4 to 8, and most preferably 4 or 5.

[0046] According to the coherent anti-Stokes Raman microscope system of the first aspect of the present invention, the pulse stretching and amplifying module comprises a first optical path and a second optical path, wherein the first optical path and the second optical path respectively and sequentially comprise: a fiber stretcher, a pulse stretching and amplifying pump laser source, a pulse stretching and amplifying wavelength division multiplexer, a pulse stretching and amplifying fiber amplifier, a fiber collimator, a pulse stretching and amplifying half-wave plate, a spatial isolator and a pulse stretching and amplifying reflector;

[0047] Preferably, the number of the pulse stretching and amplifying mirrors is 1 to 4, more preferably 2 to 3, most preferably 2; and / or

[0048] Preferably, the following are arranged in sequence between the pulse stretching and amplifying optical fiber amplifier and the optical fiber collimator:

[0049] 1) 1 to 3 groups, more preferably 1 to 2 groups, most preferably 2 groups of the pulse stretching and amplifying pump laser sources,

[0050] 2) Fiber combiner, and

[0051] 3) The pulse stretching and amplifying optical fiber amplifier.

[0052] According to the coherent anti-Stokes Raman microscope system of the first aspect of the present invention, the spectrum broadening filter module comprises in sequence: a first filter half-wave plate, a filter grating pair, a second filter half-wave plate, a polarization beam splitter, a light baffle, a filter focusing lens, an optical fiber, a filter collimating lens and a filter;

[0053] Preferably, the filter grating pair comprises a filter reflector pair and a filter transmission grating pair; and / or

[0054] Preferably, the optical fiber is selected from one or more of the following: LEAF optical fiber, photonic crystal optical fiber, high nonlinear optical fiber, more preferably LEAF optical fiber or photonic crystal optical fiber, most preferably LEAF optical fiber.

[0055] The coherent anti-Stokes Raman microscope system according to the first aspect of the present invention, wherein:

[0056] The double frequency generation module comprises in sequence: a double frequency half wave plate, a double frequency focusing lens, a double frequency crystal and a double frequency collimating lens; wherein the double frequency crystal is selected from one or more of the following: barium metaborate crystal, periodically poled lithium niobate crystal, lithium iodate, more preferably barium metaborate crystal or periodically poled lithium niobate crystal, most preferably periodically poled lithium niobate crystal; and / or

[0057] The microscope in the microscope module is selected from one or more of the following: a multiphoton microscope, a nonlinear optical microscope, a confocal microscope, preferably a multiphoton microscope or a nonlinear optical microscope, most preferably a multiphoton microscope;

[0058] Preferably, the femtosecond laser front-end module outputs a femtosecond pulse sequence, which is divided into a first optical path and a second optical path after passing through the dual-wavelength module and the pulse stretching and amplification module in sequence. The pulse output by the first optical path enters the spectral stretching filter module, is adjusted to P polarization through the first filter half-wave plate, and then is focused onto the optical fiber through a filter focusing lens for spectral broadening, and then is collimated through a filter collimating lens to output the spectrally broadened pulse, and then enters the double frequency generation module; the pulse output by the second optical path enters the time delay module.

[0059] According to the coherent anti-Stokes Raman microscope system of the first aspect of the present invention, the dispersion compensation module comprises: a dispersion compensation device group, a dispersion compensation reflector and a dichroic mirror; wherein:

[0060] The dispersion compensation component group includes: a dispersion compensation grating pair, a grating reflector pair and a dispersion compensation half-wave plate;

[0061] The dispersion compensating grating pair is a dispersion compensating transmission grating pair; and / or

[0062] The number of the dispersion compensation device groups is at least 2;

[0063] Preferably, the two pulses output by the double frequency generation module and the time delay module are respectively broadened by the dispersion compensation module, and then spatially overlapped by the dispersion compensation module, more preferably by the dichroic mirror, and then introduced into the microscope module together.

[0064] The coherent anti-Stokes Raman microscope system according to the first aspect of the present invention, wherein:

[0065] The reflectors in the optical path compensation reflector, pulse broadening and magnification reflector, dispersion compensation reflector, grating reflector pair and / or filter reflector pair are all selected from one or more of the following: plane reflector, square reflector, ultraviolet fused quartz reflector, microcrystalline glass reflector, silver-coated reflector, gold-coated reflector, preferably selected from one or more of the following: plane reflector, square reflector, silver-coated reflector, more preferably plane reflector or square reflector;

[0066] The dual-wavelength pump laser source and / or the impulse broadening and amplification pump laser source are selected from one or more of the following: a 1W diode pump laser source, a 9W diode pump laser source, a 27W diode pump laser source, more preferably a 1W diode pump laser source or a 27W diode pump laser source; and / or

[0067] The dual-wavelength fiber amplifier and / or pulse broadening and amplification fiber amplifier are selected from one or more of the following: Yb-doped single-mode fiber amplifier, Yb-doped multi-mode fiber amplifier, Er-doped single-mode fiber amplifier, Er-doped multi-mode fiber amplifier, Yb / Er co-doped multi-mode fiber amplifier; wherein:

[0068] Preferably, the Yb-doped single-mode fiber amplifier is a Yb401 single-mode fiber amplifier;

[0069] Preferably, the Yb-doped multimode fiber amplifier is a Yb12 / 125 multimode fiber amplifier and / or a Yb30 / 250 multimode fiber amplifier;

[0070] Preferably, the Er-doped single-mode fiber amplifier is an Er80 single-mode fiber amplifier and / or an Er40 single-mode fiber amplifier; and / or

[0071] Preferably, the Yb / Er co-doped multimode fiber amplifier is a Yb / Er30 / 250 multimode fiber amplifier.

[0072] A second aspect of the present invention provides a method for driving a microscope, the method comprising: using the coherent anti-Stokes Raman microscope system described in the first aspect to drive the microscope;

[0073] Preferably, the method comprises: the femtosecond laser front end emits an ultrashort pulse sequence, which is spectrally broadened by the dual-wavelength module, and then two ultrashort pulse sequences are outputted with power amplification by the pulse broadening and amplification module; one of the power-amplified ultrashort pulse sequences is adjusted for time delay by the time delay module, and the other is adjusted for wavelength by the spectral broadening and filtering module, and then converted in wavelength by the double frequency generation module, and input together with the pulse output by the time delay module into the dispersion compensation module for broadening, and then collimated into the microscope module to realize imaging of the sample.

[0074] The third aspect of the present invention provides a nonlinear optical microscope device, which includes the coherent anti-Stokes Raman microscope system described in the first aspect. Preferably, the nonlinear optical microscope device is a self-phase modulation spectral selectively driven microscope.

[0075] According to a specific embodiment of the present invention, the first aspect of the present invention provides a dual-wavelength self-phase modulation spectral selection driven CARS microscope system, the microscope system comprises: a femtosecond laser front-end module, a dual-wavelength module, a pulse broadening and amplification module, a time delay module, a spectrum broadening and filtering module, a double frequency generation module, a dispersion compensation module, and a microscope module; wherein,

[0076] The output end of the femtosecond laser front-end module is connected to the input end of the pulse dual-wavelength module, the output end of the dual-wavelength module is connected to the input end of the pulse stretching and amplification module, the output end of the pulse stretching and amplification module is connected to the input ends of the spectrum stretching and filtering module and the time delay module, the output end of the spectrum stretching and filtering module is connected to the input end of the double frequency generation module, the output ends of the double frequency generation module and the time delay module are connected to the input end of the dispersion compensation module, and the output end of the dispersion compensation module is connected to the input end of the microscope module;

[0077] Preferably, the femtosecond laser front-end module is used to output an ultrashort pulse sequence, the dual-wavelength module is used to broaden the input pulse spectrum to the corresponding band of the amplifier, the pulse stretching and amplification module is used to introduce dispersion into the input pulse to stretch the pulse width and then perform power amplification, the spectrum stretching and filtering module is used to compress the pulse width of the pulse output by the pulse stretching and amplification module, then use the self-phase modulation effect to broaden the spectrum and then filter the required band, the time delay module is used to compensate for the optical path difference of the two pulses, the double frequency generation module is used to double the frequency of the pulses output from the output end of the spectrum stretching and filtering module, the dispersion compensation module is used to compensate for the dispersion time domain broadening of the two pulse lights, and the multiphoton microscope module is driven by the pulse output by the dispersion compensation module to image the sample.

[0078] The femtosecond laser front-end module includes an oscillator; wherein,

[0079] The repetition frequency of the femtosecond pulse sequence output by the femtosecond laser front-end module is 37 MHz;

[0080] The central wavelength range of the ultrashort pulse sequence emitted by the oscillator in the femtosecond laser front-end module is 1.54 μm to 1.57 μm, and most preferably 1.55 μm;

[0081] The oscillator is a fiber laser oscillator or a solid-state laser oscillator, preferably an erbium-doped fiber laser oscillator, and more preferably a mode-locked erbium-doped fiber laser oscillator;

[0082] Preferably, the mode locking method is selected from one or more of the following: semiconductor saturable absorber mirror, nonlinear polarization rotation, nonlinear optical loop mirror, and most preferably semiconductor saturable absorber mirror;

[0083] Further preferably, the mode-locked erbium-doped fiber laser oscillator is an erbium-doped fiber laser oscillator mode-locked based on a semiconductor saturable absorber mirror.

[0084] The dual-wavelength module includes an optical fiber isolator, a diode pumped laser source, a wavelength division multiplexer, an optical fiber amplifier, and a highly nonlinear optical fiber.

[0085] Preferably, the pulse stretching and amplification module further includes a fiber stretcher, a pump laser source, a wavelength division multiplexer, a fiber amplifier, a collimator, and a half-wave plate; and / or

[0086] Preferably, the ultrashort pulse sequence emitted by the femtosecond laser front-end module is amplified to an average pulse power of 5 to 7 W, more preferably 5 to 6 W, after passing through a fiber stretcher and a fiber amplifier.

[0087] The pulse time delay module includes an optical path compensation device; preferably, the optical path compensation device is a reflector and a translation stage or a window plate, and more preferably a reflector and a translation stage.

[0088] The pulse sequence output by the pulse stretching and amplification module is directed unobstructed to the dispersion compensation device, which includes a transmission grating pair and a reflector. After being turned back by the second plane reflector and lowered in height, it passes through the dispersion compensation device again and reflects on the first plane reflector to output the compressed pulse.

[0089] The spectrum broadening and filtering module comprises: a reflector, a half-wave plate, a light baffle, a polarization beam splitter, an aspheric lens, a spectrum sidelobe filtering optical fiber and an optical filter;

[0090] Preferably, the spectral sidelobe filtering optical fiber is a large mode area photonic crystal fiber or a single mode photonic crystal fiber, further preferably a large mode area photonic crystal fiber-8 (LMA-8) or a single mode photonic crystal fiber-12 (ESM-12), and most preferably a single mode photonic crystal fiber-12 (ESM-12);

[0091] More preferably, the length of the single-mode photonic crystal fiber-12 is 5 to 10 cm, further preferably 5 to 7 cm, and most preferably 7 cm.

[0092] The double frequency generation module includes: a half-wave plate, a spherical lens, and a frequency doubling crystal; preferably, the frequency doubling crystal is BaB2O4 (BBO) or periodically poled lithium niobate (PPLN), and more preferably is a periodically poled lithium niobate (PPLN) crystal with higher frequency doubling efficiency.

[0093] The dispersion compensation module includes: a reflector, a half-wave plate, a dispersion compensation device, and a dichroic mirror. Preferably, the dispersion compensation device is a projection grating pair or a window plate, and more preferably a projection grating pair with adjustable dispersion compensation.

[0094] The microscope module comprises: a half-wave plate, a light blocking plate, a polarization beam splitter and a multi-photon microscope.

[0095] The second aspect of the present invention provides a method for driving a microscope, the method comprising: using the coherent anti-Stokes Raman microscope system driven by the self-phase modulation spectral selection of the first aspect to drive the microscope, the method comprising: the femtosecond laser front end of the microscope system emits an ultrashort pulse sequence, which is spectrally broadened by the dual-wavelength module, and the pulse broadening and amplification module outputs a power-amplified ultrashort pulse sequence, the power-amplified ultrashort pulse sequence is respectively adjusted for time delay by a time delay module, and after wavelength conversion by a spectral broadening and filtering module and a double frequency generation module, is input into a dispersion compensation module for broadening, and then collimated to enter the microscope, and finally the imaging of the sample is realized by the microscope module.

[0096] A third aspect of the present invention provides a nonlinear optical microscope, which includes the self-phase modulation spectrally selectively driven coherent anti-Stokes Raman microscope system described in the first aspect.

[0097] According to another specific embodiment of the present invention, in a coherent anti-Stokes Raman microscope system driven by self-phase modulation spectral selection of the present invention, the dual-wavelength module is used to broaden the 1.55um pulse spectrum to 1.03um, the time delay module is used to compensate the optical path of the Stokes light so that the two columns of pulses overlap in time, the spectrum broadening and filtering module is used to compress one column of pulses output by the dual-wavelength module, broaden the spectrum by using the self-phase modulation effect, and then filter the required band, the dispersion compensation module is used to broaden the pulses output from the output ends of the double frequency generation module and the time delay module to improve the spectral resolution, and the two columns of pulses output by the dispersion compensation module are collimated by a dichroic mirror and enter the microscope module to image the sample.

[0098] The central wavelength range of the ultrashort pulse sequence emitted by the oscillator in the femtosecond laser front-end module is 1.5 μm to 1.55 μm, and the most preferred wavelength is 1.55 μm;

[0099] The dual-wavelength module includes a fiber isolator, a fiber amplifier and a highly nonlinear fiber;

[0100] Preferably, the pulse stretching and amplification module further includes a pump laser source, a wavelength division multiplexer, and a collimator; and / or

[0101] Preferably, the spectrum of the ultrashort pulse sequence emitted by the femtosecond laser front-end module is broadened to 1.03 um after passing through the highly nonlinear optical fiber.

[0102] The pulse stretching and amplification module includes a fiber isolator, a fiber stretcher and a two-stage fiber amplifier;

[0103] Preferably, the pulse stretching and amplification module further includes a pump laser source, a wavelength division multiplexer, and a collimator; and / or

[0104] Preferably, the ultrashort pulse sequence emitted by the femtosecond laser front-end module is amplified to an average pulse power of 5 to 7 W, more preferably 5 to 6 W, after passing through a fiber stretcher and a fiber amplifier.

[0105] The pulse time delay module comprises a plane reflector and an adjustable translation stage. The plane reflector is mounted on the adjustable translation stage. The size of the compensation optical path is adjusted by moving the translation stage.

[0106] The pulse sequence output by the pulse stretching and amplification module is directed unimpeded toward the dispersion compensation device of the pulse compression module, then returns through the second plane reflector and reduces its height before passing through the dispersion compensation device again, and is reflected on the first plane reflector to output a compressed pulse.

[0107] The spectrum broadening and filtering module comprises: a reflector, a half-wave plate, a dispersion compensation device, a light baffle, a polarization beam splitter, an aspheric lens, a spectrum sidelobe filtering optical fiber and an optical filter.

[0108] The pulse sequence output by the pulse stretching and amplification module is directed to the dispersion compensation device without obstruction, then returns through the second plane reflector and reduces its height before passing through the dispersion compensation device again, and is reflected on the first plane reflector to output a compressed pulse.

[0109] The two pulses emitted by the double frequency generation module and the time delay module are respectively broadened by the transmission grating pair, collimated by the dichroic mirror to achieve spatial overlap, and then introduced into the microscope module.

[0110] The reflector is selected from one or more of the following: a plane reflector, a square reflector, an ultraviolet fused quartz reflector, a microcrystalline glass reflector, a silver-coated reflector, and a gold-coated reflector, preferably selected from one or more of the following: a plane reflector, a square reflector, and a silver-coated reflector, and more preferably a plane reflector or a square reflector;

[0111] The coherent anti-Stokes Raman microscope system based on self-phase modulation spectral selection drive of the present invention can obtain 800nm ​​pump light and 1030nm Stokes light from a 1550nm laser neutron source to achieve coherent anti-Stokes Raman (CARS) microscopic imaging, that is, to achieve selective synchronous imaging of multiple biological tissue components. At the same time, the microscope system based on self-phase modulation spectral selection drive of the present invention has strong scalability, and the wavelength of the light source can be tuned by adjusting the size of the input optical fiber energy to suit the selective imaging of components in different samples.

[0112] The coherent anti-Stokes Raman microscope system driven by self-phase modulation spectral selection of the present invention can have but is not limited to the following beneficial effects:

[0113] 1. The coherent anti-Stokes Raman microscope system driven by self-phase modulation spectral selection of the present invention adopts the method of self-phase modulation spectral selection (SESS) to convert the pulse wavelength of 1550nm to 1600nm, realizes the second harmonic generation process through the frequency doubling crystal, converts the pulse wavelength of 1600nm to 800nm, realizes coherent anti-Stokes Raman microscopic imaging, that is, realizes selective imaging of various biological tissue components. Compared with other wavelength conversion methods, the device is simple, the conversion efficiency is high, and the wavelength is tunable.

[0114] 2. Strong scalability: the self-phase modulation spectral selection driven microscope system of the present invention can tune the wavelength of the light source by adjusting the input optical fiber energy to suit the imaging of different samples. BRIEF DESCRIPTION OF THE DRAWINGS

[0115] The embodiments of the present invention are described in detail below with reference to the accompanying drawings, wherein:

[0116] Figure 1 A schematic structural diagram of a self-phase modulation spectral selection driven CARS microscope system of Example 1 is shown.

[0117] Explanation of the accompanying drawings: 1. Femtosecond laser front-end module; 2. Dual-wavelength module; 3. Pulse stretching and amplification module; 4. Time delay module; 5. Spectral stretching and filtering module; 6. Double frequency generation module; 7. Dispersion compensation module; 8. Microscope module; 9. Dual-wavelength optical fiber isolator; 10. Dual-wavelength pump laser source; 11. Dual-wavelength wavelength division multiplexer; 12. Dual-wavelength optical fiber amplifier; 13. High nonlinear optical fiber; 14. First optical fiber stretcher; 15. Second optical fiber stretcher; 16. First pulse stretching and amplifying pump laser source; 17. Second pulse stretching and amplifying pump laser source; 18. First pulse stretching and amplifying wavelength division multiplexer ; 19. Second pulse stretching and amplifying wavelength division multiplexer; 20. First pulse stretching and amplifying fiber amplifier; 21. Second pulse stretching and amplifying fiber amplifier; 22. Third pulse stretching and amplifying pump laser source; 23. Fourth pulse stretching and amplifying pump laser source; 24. First fiber combiner; 25. Second fiber combiner; 26. Third pulse stretching and amplifying fiber amplifier; 27. Fourth pulse stretching and amplifying fiber amplifier; 28. First fiber collimator; 29. ​​Second fiber collimator; 30. First pulse stretching and amplifying half wave plate; 31. Second pulse stretching and amplifying half wave plate; 32. First spatial isolator; 33 , the second spatial isolator; 34, the first pulse stretching and magnifying reflector; 35, the first optical path compensation reflector; 36, the second optical path compensation reflector; 37, the third optical path compensation reflector; 38, the fourth optical path compensation reflector; 39, the fifth optical path compensation reflector; 40, the first dispersion compensation half wave plate; 41, the first square reflector; 42, the first dispersion compensation transmission grating; 43, the second dispersion compensation transmission grating; 44, the first grating reflector; 45, the dispersion compensation reflector; 46, the second pulse stretching and magnifying reflector; 47, the first filtering half wave plate; 48, the first filtering reflector; 49, the first filtering Transmission grating; 50, second filter transmission grating; 51, second filter reflector; 52, second filter half wave plate; 53, polarization beam splitter; 54, light baffle; 55, filter focusing lens; 56, optical fiber; 57, filter collimating lens; 58, filter; 59, double frequency half wave plate; 60, double frequency focusing lens; 61, frequency doubling crystal; 62, double frequency collimating lens; 63, second dispersion compensating half wave plate; 64, second square reflector; 65, third dispersion compensating transmission grating; 66, fourth dispersion compensating transmission grating; 67, second grating reflector; 68, dichroic mirror; 69, microscope.

[0118] A. Ultrashort pulse sequence emitted by femtosecond laser front-end module 1; B. Pulse A is amplified and spectrally broadened into 1um and 1.5um wavelength pulses through nonlinear fiber optic spectral broadening; C. Pulse B is a dual-wavelength pulse after chirped pulse amplification by module 3; D. Pulse output by spectral broadening filter module 5; E. Pulse D is a pump pulse output by frequency doubling generation module 6; F. Pump pulse and Stokes pulse after temporal and spatial overlap. DETAILED DESCRIPTION

[0119] The present invention will be further described below through specific embodiments in conjunction with the accompanying drawings. However, it should be understood that these embodiments are only used for more detailed and specific description and should not be understood as limiting the present invention in any form.

[0120] Example 1

[0121] This embodiment is used to illustrate the structure of the coherent anti-Stokes Raman microscope system driven by self-phase modulation spectral selection of the present invention.

[0122] Figure 1 The invention is a coherent anti-Stokes Raman microscope system based on self-phase modulation spectral selection drive. It includes: a femtosecond laser front-end module 1; a dual-wavelength module 2; a pulse broadening and amplification module 3; a time delay module 4; a spectrum broadening and filtering module 5; a double frequency generation module 6; a dispersion compensation module 7; and a microscope module 8. In this embodiment, the femtosecond laser front-end module 1 adopts an ytterbium-doped fiber oscillator with semiconductor protected absorption mirror mode locking, and the parameters corresponding to the output ultrashort pulse sequence A are: a central wavelength of 1.55 μm and a repetition frequency of 37 MHz.

[0123] After the output ultrashort pulse sequence A is amplified by the dual-wavelength fiber amplifier 12 in the dual-wavelength module 2 and the spectrum is broadened by the high nonlinear fiber 13, the output spectrum includes 1.03um and 1.55um pulses B. Pulse B is divided into two paths and respectively amplified by the first fiber stretcher 14 and the second fiber stretcher 15, and then amplified by the first pulse broadening and amplifying fiber amplifier 20, the second pulse broadening and amplifying fiber amplifier 21, the third pulse broadening and amplifying fiber amplifier 26, and the fourth pulse broadening and amplifying fiber amplifier 27 to output pulse C, which includes high-power 1.03um pulses and 1.55um pulses.

[0124] The pulse with a wavelength of 1.55um in pulse C is incident on a grating pair consisting of a first filter transmission grating 49 and a second filter transmission grating 50. The first filter transmission grating 49 and the second filter transmission grating 50 are placed in parallel, and the second filter transmission grating 50 is mounted on a precision adjustable displacement platform, which can flexibly control the spacing between the grating pairs. The pulse sequence passes through the second filter reflector 51, turns downward, lowers a certain height, and then returns again and passes through the grating pair, and finally reflects at an angle of 45 degrees on the first filter reflector 48 to output a compressed pulse.

[0125] The compressed pulse is adjusted to P polarization through the second filtering half wave plate 52 and the polarization beam splitter 53, and then the pulse is focused onto the core of the LEAF optical fiber 56 through the filtering focusing lens 55. The pulse spectrum is broadened by the LEAF optical fiber 56, and then collimated by the filtering collimating lens 57, and then the required spectral part, namely the output pulse D, is filtered out by the filter 58.

[0126] Pulse D is adjusted to P polarization by the double frequency half wave plate 59, and then the pulse is focused onto the double frequency crystal 61 through the double frequency focusing lens 60 to generate the second harmonic, and then is collimated by the double frequency collimating lens 62 to output pulse E.

[0127] The pulse with a wavelength of 1.03um in pulse C enters the time delay module and is reflected on the second optical path compensation reflector 36, the third optical path compensation reflector 37, the fourth optical path compensation reflector 38, and the fifth optical path compensation reflector 39. The third optical path compensation reflector 37 and the fourth optical path compensation reflector 38 are mounted on the same precision adjustable displacement platform, and the optical path of the 1.03um pulse can be adjusted within a certain range to ensure time overlap with the 1.55um pulse. Then pulse E is output.

[0128] The two pulses of pulse E are adjusted to P polarization through the first dispersion compensation half wave plate 40 and the second dispersion compensation half wave plate 63, and then compensate the second-order dispersion through the first dispersion compensation transmission grating 42, the second dispersion compensation transmission grating 43, the third dispersion compensation transmission grating 65, and the fourth dispersion compensation transmission grating 66. Each pulse E is widened to about 3ps. Afterwards, the two light beams are spatially overlapped through the dichroic mirror 68, and the pulse F is output and incident on the microscope 69 to realize the imaging of the biological sample.

[0129] Example 2

[0130] This embodiment is used to illustrate the wavelength conversion of the coherent anti-Stokes Raman microscope system driven by self-phase modulation spectral selection of the present invention.

[0131] This embodiment uses nonlinear spectral broadening, self-phase modulation spectral selection (SESS) and second harmonic generation (SHG) methods to generate pump light and Stokes light for driving CARS imaging.

[0132] The 1030nm pulse spectrum is broadened to 1550nm by the nonlinear effect in the highly nonlinear optical fiber 13, and is amplified by the ytterbium-doped and erbium-doped optical fibers respectively, to obtain two pulses with wavelengths of 1030nm and 1550nm.

[0133] After the pulse with a wavelength of 1550nm is compressed by the first filter transmission grating 49 and the second filter transmission grating 50, the pulse of 1550nm is focused into the LEAF optical fiber 56 through the filter focusing lens 55. The high-energy narrow-band femtosecond pulse is transmitted in the optical fiber with a length of 7.5±2.5cm to obtain sufficient spectrum broadening. The broadened spectrum is composed of a series of independent spectrum side lobes, in which the leftmost and rightmost side lobes contain a large amount of pulse energy. The light transmitted in the LEAF optical fiber 56 is output as collimated light through the filter collimating lens 57, and the leftmost / rightmost spectrum side lobes containing the most energy are filtered out by the filter 58, so that the near-conversion limit femtosecond pulse with a wide range of tunable wavelength can be obtained to achieve wavelength conversion. When the rightmost side of the spectrum is broadened to 1600nm, the right side lobe is filtered out by the filter 58, that is, the output pulse D.

[0134] Pulse D is adjusted to P polarization through the double frequency half wave plate 59, and then the 1600nm pulse is focused onto the frequency doubling crystal 61 through the double frequency focusing lens 60. The high-energy narrow-band femtosecond pulse generates a second harmonic with a wavelength of 800nm ​​on the frequency doubling crystal 61. After the wavelength conversion is completed, the collimated output pulse E is output through the double frequency collimating lens 62.

[0135] The pulses with a wavelength of 800 nm and 1.03 μm are introduced into the dispersion compensation module 7 by compensating the second-order dispersion through the first dispersion compensation transmission grating 42, the second dispersion compensation transmission grating 43, the third dispersion compensation transmission grating 65, and the fourth dispersion compensation transmission grating 66 to increase the spectral resolution. Then, the pulses are spatially overlapped through the dichroic mirror 68 and introduced into the microscope 69.

[0136] Using pulse F to drive the microscope 69 can stimulate stronger signals and collect signals through the C—H bonds in the coherent anti-Stokes Raman imaging sample. The present invention can easily achieve wavelength conversion and obtain selective imaging suitable for coherent anti-Stokes Raman microscopy.

[0137] Although only a specific embodiment and its effects are shown above, those skilled in the art should understand that according to the concept of the present invention, the aforementioned other embodiments without specific effects or other technical solutions of the present invention not shown in the embodiments can also achieve the following technical effects stated in the invention content section that are equivalent to the embodiments:

[0138] 1. The coherent anti-Stokes Raman microscope system driven by self-phase modulation spectral selection of the present invention adopts the method of self-phase modulation spectral selection (SESS) to convert the pulse wavelength of 1550nm to 1600nm, realizes the second harmonic generation process through the frequency doubling crystal, converts the pulse wavelength of 1600nm to 800nm, realizes coherent anti-Stokes Raman microscopic imaging, that is, realizes selective imaging of various biological tissue components. Compared with other wavelength conversion methods, the device is simple, the conversion efficiency is high, and the wavelength is tunable.

[0139] 2. Strong scalability: the self-phase modulation spectral selection driven microscope system of the present invention can tune the wavelength of the light source by adjusting the input optical fiber energy to suit the imaging of different samples.

[0140] Although the present invention has been described to a certain extent, it is obvious that appropriate changes in various conditions can be made without departing from the spirit and scope of the present invention. It is understood that the present invention is not limited to the embodiments described, but belongs to the scope of the claims, which includes equivalent replacements of each factor described.

Claims

1. A coherent anti-Stokes Raman microscope system, characterized in that: The coherent anti-Stokes Raman microscope system comprises: Femtosecond laser front-end module for outputting ultrashort pulse trains, A dual-wavelength module for broadening the spectrum of the ultrashort pulse sequence, A pulse stretching and amplification module is used to introduce dispersion into the stretched pulse to stretch the pulse width and then amplify the power. Time delay module used to compensate for the optical path difference of two pulses. A spectrum broadening filter module is used to filter the spectrum after broadening the pulse width output by the pulse broadening and amplification module, A frequency doubling generation module for doubling the frequency of the pulse output from the spectrum broadening and filtering module, A dispersion compensation module for compensating the dispersion time-domain broadening of two pulsed optical beams, and Microscope module; Furthermore, the output end of the femtosecond laser front-end module is connected to the input end of the dual-wavelength module, the output end of the dual-wavelength module is connected to the input end of the pulse stretching and amplification module, the output end of the pulse stretching and amplification module is connected to the input end of the time delay module and the spectrum stretching and filtering module, the output end of the spectrum stretching and filtering module is connected to the input end of the double frequency generation module, the output ends of the double frequency generation module and the time delay module are connected to the input end of the dispersion compensation module, and the output end of the dispersion compensation module is connected to the input end of the microscope module; Preferably, the spectrum broadening of the spectrum broadening filter module is achieved through a self-phase modulation effect, and / or Preferably, the multiphoton microscope module is driven by pulses output by the dispersion compensation module to image the sample.

2. The coherent anti-Stokes Raman microscope system according to claim 1, characterized in that: The femtosecond laser front-end module includes an oscillator; wherein, The repetition frequency of the femtosecond pulse sequence output by the femtosecond laser front-end module is 35-40 MHz, and most preferably 37 MHz; The central wavelength range of the ultrashort pulse sequence emitted by the oscillator in the femtosecond laser front-end module is 1.50 μm to 1.66 μm, preferably 1.54 μm to 1.57 μm, and most preferably 1.55 μm; and / or The oscillator is a fiber laser oscillator or a solid-state laser oscillator, preferably an erbium-doped fiber laser oscillator, more preferably a mode-locked erbium-doped fiber laser oscillator; Preferably, the mode locking method is selected from one or more of the following: semiconductor saturable absorber mirror, nonlinear polarization rotation, nonlinear optical loop mirror, and most preferably semiconductor saturable absorber mirror.

3. The coherent anti-Stokes Raman microscope system according to claim 1 or 2, characterized in that: The dual-wavelength module includes in sequence: a dual-wavelength optical fiber isolator, a dual-wavelength pump laser source, a dual-wavelength wavelength division multiplexer, a dual-wavelength optical fiber amplifier and a high nonlinear optical fiber; and / or The time delay module includes an optical path compensation device; wherein the optical path compensation device includes: an optical path compensation reflector and an optical path compensation translation stage; Preferably, the optical path compensation reflector is mounted on the optical path compensation translation stage; and / or Preferably, the number of the optical path compensation reflectors is 2 to 10, more preferably 4 to 8, and most preferably 4 or 5.

4. The coherent anti-Stokes Raman microscope system according to any one of claims 1 to 3, characterized in that: The pulse stretching and amplification module comprises a first optical path and a second optical path, wherein the first optical path and the second optical path respectively comprise: an optical fiber stretcher, a pulse stretching and amplification pump laser source, a pulse stretching and amplification wavelength division multiplexer, a pulse stretching and amplification optical fiber amplifier, an optical fiber collimator, a pulse stretching and amplification half-wave plate, a spatial isolator and a pulse stretching and amplification reflector; Preferably, the number of the pulse stretching and amplifying mirrors is 1 to 4, more preferably 2 to 3, most preferably 2; and / or Preferably, the following are arranged in sequence between the pulse stretching and amplifying optical fiber amplifier and the optical fiber collimator: 1) 1 to 3 groups, more preferably 1 to 2 groups, most preferably 2 groups of the pulse stretching and amplifying pump laser sources, 2) Fiber combiner, and 3) The pulse stretching and amplifying optical fiber amplifier.

5. The coherent anti-Stokes Raman microscope system according to any one of claims 1 to 4, characterized in that: The spectrum broadening filter module includes in sequence: a first filter half wave plate, a filter grating pair, a second filter half wave plate, a polarization beam splitter, a light baffle, a filter focusing lens, an optical fiber, a filter collimating lens and a filter; Preferably, the filter grating pair comprises a filter reflector pair and a filter transmission grating pair; and / or Preferably, the optical fiber is selected from one or more of the following: LEAF optical fiber, photonic crystal optical fiber, high nonlinear optical fiber, more preferably LEAF optical fiber or photonic crystal optical fiber, most preferably LEAF optical fiber.

6. The coherent anti-Stokes Raman microscope system according to any one of claims 1 to 5, characterized in that: The double frequency generation module comprises in sequence: a double frequency half wave plate, a double frequency focusing lens, a double frequency crystal and a double frequency collimating lens; wherein the double frequency crystal is selected from one or more of the following: barium metaborate crystal, periodically poled lithium niobate crystal, lithium iodate, more preferably barium metaborate crystal or periodically poled lithium niobate crystal, most preferably periodically poled lithium niobate crystal; and / or The microscope in the microscope module is selected from one or more of the following: a multiphoton microscope, a nonlinear optical microscope, a confocal microscope, preferably a multiphoton microscope or a nonlinear optical microscope, most preferably a multiphoton microscope; Preferably, the femtosecond laser front-end module outputs a femtosecond pulse sequence, which is divided into a first optical path and a second optical path after passing through the dual-wavelength module and the pulse stretching and amplification module in sequence. The pulse output by the first optical path enters the spectral stretching filter module, is adjusted to P polarization through the first filter half-wave plate, and then is focused onto the optical fiber through a filter focusing lens for spectral broadening, and then is collimated through a filter collimating lens to output the spectrally broadened pulse, and then enters the double frequency generation module; the pulse output by the second optical path enters the time delay module.

7. The coherent anti-Stokes Raman microscope system according to any one of claims 1 to 6, characterized in that: The dispersion compensation module comprises: a dispersion compensation device group, a dispersion compensation reflector and a dichroic mirror; wherein: The dispersion compensation component group includes: a dispersion compensation grating pair, a grating reflector pair and a dispersion compensation half-wave plate; The dispersion compensating grating pair is a dispersion compensating transmission grating pair; and / or The number of the dispersion compensation device groups is at least 2; Preferably, the two pulses output by the double frequency generation module and the time delay module are respectively broadened by the dispersion compensation module, and then spatially overlapped by the dispersion compensation module, more preferably by the dichroic mirror, and then introduced into the microscope module together.

8. The coherent anti-Stokes Raman microscope system according to claim 3 or 4, characterized in that: The reflectors in the optical path compensation reflector, pulse broadening and magnification reflector, dispersion compensation reflector, grating reflector pair and / or filter reflector pair are all selected from one or more of the following: plane reflector, square reflector, ultraviolet fused quartz reflector, microcrystalline glass reflector, silver-coated reflector, gold-coated reflector, preferably selected from one or more of the following: plane reflector, square reflector, silver-coated reflector, more preferably plane reflector or square reflector; The dual-wavelength pump laser source and / or the impulse broadening and amplification pump laser source are selected from one or more of the following: a 1W diode pump laser source, a 9W diode pump laser source, a 27W diode pump laser source, more preferably a 1W diode pump laser source or a 27W diode pump laser source; and / or The dual-wavelength fiber amplifier and / or pulse broadening and amplification fiber amplifier are selected from one or more of the following: Yb-doped single-mode fiber amplifier, Yb-doped multi-mode fiber amplifier, Er-doped single-mode fiber amplifier, Er-doped multi-mode fiber amplifier, Yb / Er co-doped multi-mode fiber amplifier; wherein: Preferably, the Yb-doped single-mode fiber amplifier is a Yb401 single-mode fiber amplifier; Preferably, the Yb-doped multimode fiber amplifier is a Yb12 / 125 multimode fiber amplifier and / or a Yb30 / 250 multimode fiber amplifier; Preferably, the Er-doped single-mode fiber amplifier is an Er80 single-mode fiber amplifier and / or an Er40 single-mode fiber amplifier; and / or Preferably, the Yb / Er co-doped multimode fiber amplifier is a Yb / Er30 / 250 multimode fiber amplifier.

9. A method for driving a microscope, characterized in that: The method comprises: using the coherent anti-Stokes Raman microscope system according to any one of claims 1 to 8 to drive a microscope; Preferably, the method comprises: the femtosecond laser front end emits an ultrashort pulse sequence, which is spectrally broadened by the dual-wavelength module, and then two ultrashort pulse sequences are outputted with power amplification by the pulse broadening and amplification module; one of the power-amplified ultrashort pulse sequences is adjusted for time delay by the time delay module, and the other is adjusted for wavelength by the spectral broadening and filtering module, and then converted in wavelength by the double frequency generation module, and input together with the pulse output by the time delay module into the dispersion compensation module for broadening, and then collimated into the microscope module to realize imaging of the sample.

10. A nonlinear optical microscope device, characterized in that: The nonlinear optical microscope device comprises the coherent anti-Stokes Raman microscope system according to any one of claims 1 to 8. Preferably, the nonlinear optical microscope device is a self-phase modulation spectral selectively driven microscope.

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