Coherent anti-stokes raman microscopy system, its driving method and microscopy apparatus

Abstract

This invention provides a coherent anti-Stokes Raman microscope system, its driving method, and its microscopic apparatus. The coherent anti-Stokes Raman microscope system includes: a femtosecond laser front-end module, a dual-wavelength module, a pulse broadening and amplification module, a time delay module, a spectral broadening and filtering module, a second harmonic generation module, a dispersion compensation module, and a microscope module. The self-phase modulation spectral selection (SESS) driven coherent anti-Stokes Raman microscope system of this invention uses a self-phase modulation spectral selection (SESS) method to convert a 1550 nm pulse wavelength to 1600 nm. Through a frequency doubling crystal, a second harmonic generation process is achieved, converting the 1600 nm pulse wavelength to 800 nm, thus realizing coherent anti-Stokes Raman microscopy imaging, i.e., selective imaging of various biological tissue components. Compared with other wavelength conversion methods, the device is simple, has high conversion efficiency, is tunable, and has strong scalability.

Description

Technical Field

[0001] This invention belongs to the field of ultrafast laser technology, specifically relating to a coherent anti-Stokes Raman microscope system, its driving method, and its microscopic device. Background Technology

[0002] Coherent anti-Stokes Raman scattering (CARS) is a label-free optical microscopy imaging method based on molecular chemical bond vibrations. Due to its label-free nature, high specificity, and low optical damage, it has a wide range of applications in biological tissue imaging and pharmacokinetics.

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

[0004] To achieve selective imaging and increase the signal-to-noise ratio, a picosecond (ps) laser with tunable wavelength should be selected as the driving light source. Currently, most driving light sources are implemented using methods such as soliton self-frequency shifting, electrical modulation, and four-wave mixing, but each of these current light source implementation technologies has its own shortcomings.

[0005] Deficiencies of existing technology:

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

[0007] 2. Wavelength conversion techniques based on soliton self-frequency shift and supercontinuum spectrum have limited output pulse energy. Summary of the Invention

[0008] Therefore, the purpose of this invention is to overcome the deficiencies in the prior art and provide a coherent anti-Stokes Raman microscope system, its driving method, and the microscope apparatus. The CARS microscope system of this invention is based on a dual-wavelength system with self-phase modulation spectral selection (SESS) and a second-harmonic ultrafast fiber laser source to drive the CARS system. Compared with wavelength conversion methods 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 content of this invention, the following terms are defined as follows:

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

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

[0012] The term "ESM-12" refers to: Single-mode photonic crystal fiber-12.

[0013] The term "SESS" refers to Self-Phase Modulation Spectral Selection.

[0014] The term "P-polarization" refers to the phenomenon where, when light passes through the surface of an optical element (such as a beam splitter) at a non-perpendicular angle, both reflection and transmission characteristics depend on polarization. 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 ray lies in this plane, it is called P-polarization.

[0015] The term "LEAF fiber" refers to large effective-area fiber, a type of single-mode dispersion-shifted fiber that has a larger effective area than standard dispersion-shifted fiber, with an effective area ≥ 72µm. 2 Therefore, it has a large power handling capacity and is suitable for use with high-output-power erbium-doped fiber amplifiers.

[0016] The term "BBO" refers to barium metaborate.

[0017] The term "PPLN" refers to periodically polarized lithium niobate.

[0018] The term "Yb401 single-mode fiber amplifier" refers to 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 type of 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 objectives, a first aspect of the present invention provides a coherent anti-Stokes Raman microscopy system, the coherent anti-Stokes Raman microscopy system comprising:

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

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

[0027] A pulse widening and amplification module used to widen the pulse width by introducing dispersion into the widened pulse and then amplify its power.

[0028] Module used to compensate for the time delay of the optical path difference between two pulses.

[0029] A spectral broadening and filtering module used to broaden the spectrum of the pulse output from the pulse broadening and amplification module after pulse width compression, and then filter it.

[0030] A second harmonic generation module for multiplying the pulse output from the spectral broadening and filtering module.

[0031] A dispersion compensation module for time-domain broadening of dispersion compensation in two-channel pulsed light, and

[0032] Microscope module;

[0033] Furthermore, the output of the femtosecond laser front-end module is connected to the input of the dual-wavelength module, the output of the dual-wavelength module is connected to the input of the pulse broadening and amplification module, the output of the pulse broadening and amplification module is connected to the input of the time delay module and the spectral broadening and filtering module, the output of the spectral broadening and filtering module is connected to the input of the second harmonic generation module, the outputs of the second harmonic generation module and the time delay module are connected to the input of the dispersion compensation module, and the output of the dispersion compensation module is connected to the input of the microscope module.

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

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

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

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

[0038] The ultrashort pulse sequence emitted by the oscillator in the femtosecond laser front-end module has a center wavelength range of 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, and 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 ring mirror, and most preferably semiconductor saturable absorber mirror.

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

[0042] The dual-wavelength module sequentially comprises: a dual-wavelength fiber isolator, a dual-wavelength pump laser source, a dual-wavelength wavelength division multiplexer, a dual-wavelength fiber amplifier, and a highly nonlinear 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 mirror and an optical path compensation translation stage;

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

[0045] Preferably, the number of optical path compensation mirrors 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 amplification module includes a first optical path and a second optical path, wherein the first optical path and the second optical path respectively include, in sequence: 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;

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

[0048] Preferably, the following are further arranged sequentially between the pulse-stretching fiber amplifier and the fiber collimator:

[0049] 1) The pulse broadening and amplification pump laser source with 1 to 3 groups, more preferably 1 to 2 groups, and most preferably 2 groups,

[0050] 2) Fiber optic combiner, and

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

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

[0053] Preferably, the filter grating pair includes a filter mirror pair and a filter transmission grating pair; and / or

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

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

[0056] The second harmonic generation module sequentially includes: a second harmonic half-wave plate, a second harmonic focusing lens, a second harmonic crystal, and a second harmonic collimating lens; wherein the second harmonic crystal is selected from one or more of the following: barium metaborate crystal, periodically polarized lithium niobate crystal, lithium iodate, more preferably barium metaborate crystal or periodically polarized lithium niobate crystal, and most preferably periodically polarized lithium niobate crystal; and / or

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

[0058] Preferably, the femtosecond laser front-end module outputs a femtosecond pulse sequence, which is then divided into a first optical path and a second optical path after passing through the dual-wavelength module and the pulse broadening and amplification module. The pulses output from the first optical path enter the spectral broadening and filtering module, are adjusted to P-polarization by the first half-wave plate, are then focused onto the optical fiber by the filtering focusing lens for spectral broadening, and are then collimated by the filtering collimating lens to output the spectrally broadened pulses before entering the second harmonic generation module. The pulses output from the second optical path enter the time delay module.

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

[0060] The dispersion compensation device group includes: a dispersion compensation grating pair, a grating mirror 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 dispersion compensation device groups is at least 2;

[0063] Preferably, the two pulses output by the second harmonic generation module and the time delay module are broadened by the dispersion compensation module and then spatially overlapped by the dispersion compensation module. More preferably, they are spatially overlapped by the dichroic mirror and then imported into the microscope module together.

[0064] According to the coherent anti-Stokes Raman microscope system of 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 selected from one or more of the following: plane reflector, square reflector, ultraviolet fused silica reflector, microcrystalline glass reflector, silver-plated reflector, gold-plated reflector, preferably selected from one or more of the following: plane reflector, square reflector, silver-plated reflector, more preferably plane reflector or square reflector;

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

[0067] The dual-wavelength fiber amplifier and / or pulse-stretching fiber amplifier are each selected from one or more of the following: Yb-doped single-mode fiber amplifier, Yb-doped multimode fiber amplifier, Er-doped single-mode fiber amplifier, Er-doped multimode fiber amplifier, and Yb / Er co-doped multimode 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 includes: the femtosecond laser front end emits an ultrashort pulse sequence, which is then spectrally broadened by the dual-wavelength module, and then outputs two power-amplified ultrashort pulse sequences by the pulse broadening and amplification module. One of the power-amplified ultrashort pulse sequences is time-delayed by the time delay module, and the other is wavelength-adjusted by the spectral broadening and filtering module. The wavelength is then converted by the frequency second harmonic generation module, and the pulses output by the time delay module are input together with the pulses to the dispersion compensation module for broadening. Finally, the pulses are collimated and input into the microscope module to achieve sample imaging.

[0074] A third aspect of the present invention provides a nonlinear optical microscopy device, the nonlinear optical microscopy device comprising the coherent anti-Stokes Raman microscope system described in the first aspect, preferably, the nonlinear optical microscopy device being a self-phase-modulated spectrally selectively driven microscope.

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

[0076] The output of the femtosecond laser front-end module is connected to the input of the pulse dual-wavelength module. The output of the dual-wavelength module is connected to the input of the pulse broadening and amplification module. The output of the pulse broadening and amplification module is connected to the input of the spectral broadening and filtering module and the time delay module. The output of the spectral broadening and filtering module is connected to the input of the second harmonic generation module. The outputs of the second harmonic generation module and the time delay module are connected to the input of the dispersion compensation module. The output of the dispersion compensation module is connected to the input 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 spectrum of the input pulse to the corresponding band of the amplifier; the pulse broadening and amplification module is used to introduce dispersion into the input pulse, broaden the pulse width, and then amplify the power; the spectral broadening and filtering module is used to compress the pulse width of the pulse output by the pulse broadening and amplification module, broaden the spectrum using the self-phase modulation effect, and then filter the desired band; the time delay module is used to compensate for the optical path difference between the two pulses; the frequency doubling module is used to frequency multiply the pulse output from the output end of the spectral broadening and filtering module; the dispersion compensation module is used to compensate for the dispersion of the two pulses and broaden the time domain; 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 femtosecond laser front-end module outputs a femtosecond pulse sequence with a repetition frequency of 37MHz;

[0080] The ultrashort pulse sequence emitted by the oscillator in the femtosecond laser front-end module has a center wavelength range of 1.54μm to 1.57μm, with the most preferred value being 1.55μm;

[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 ring mirror, and most preferably semiconductor saturable absorber mirror;

[0083] More preferably, the mode-locked erbium-doped fiber laser oscillator is an erbium-doped fiber laser oscillator based on mode-locking with 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 an 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 average power of the ultrashort pulse sequence emitted by the femtosecond laser front-end module is amplified to 5-7W after passing through an optical fiber stretcher and an optical fiber amplifier, more preferably 5-6W.

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

[0088] The pulse sequence output by the pulse broadening and amplification module is directed unobstructed toward the dispersion compensation device, which includes a transmission grating pair and a reflector. After being refracted and reduced in height by a second plane reflector, the pulse passes through the dispersion compensation device again and is reflected back onto the first plane reflector, resulting in a compressed pulse output.

[0089] The spectral broadening and filtering module includes: a mirror, a half-wave plate, a light-blocking plate, a polarization beam splitter, an aspherical lens, a spectral sidelobe filtering fiber, and an optical filter;

[0090] Preferably, the spectral sidelobe filtering fiber is a large-mode-field photonic crystal fiber or a single-mode photonic crystal fiber, more preferably a large-mode-field 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, even more preferably 5 to 7 cm, and most preferably 7 cm.

[0092] The frequency doubling module includes: a half-wave plate, a spherical lens, and a frequency doubling crystal; preferably, the frequency doubling crystal is BaB2O4 (BBO) or periodically polarized lithium niobate (PPLN), and more preferably, a periodically polarized 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 pair of projection gratings or a window, and more preferably, a pair of projection gratings with adjustable dispersion compensation.

[0094] The microscope module includes: a half-wave plate, a light-blocking plate, a polarizing beam splitter, and a multiphoton microscope.

[0095] A second aspect of the present invention provides a method for driving a microscope, the method comprising: driving the microscope using a self-phase modulation spectrally selected coherent anti-Stokes Raman microscope system of the first aspect, the method comprising: emitting an ultrashort pulse sequence from a femtosecond laser front end of the microscope system, which is then spectrally broadened by a dual-wavelength module; outputting a power-amplified ultrashort pulse sequence by a pulse broadening and amplification module; the power-amplified ultrashort pulse sequence having its time delay adjusted by a time delay module; being wavelength-converted by a spectral broadening and filtering module and a second harmonic generation module; then being input into a dispersion compensation module for broadening; subsequently collimated into the microscope; and finally, imaging of the sample is achieved by the microscope module.

[0096] A third aspect of the present invention provides a nonlinear optical microscope comprising the self-phase-modulated spectral selection driven coherent anti-Stokes Raman microscope system described in the first aspect.

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

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

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

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

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

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

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

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

[0105] The pulse time delay module includes a plane mirror and an adjustable translation stage. The plane mirror is mounted on an adjustable translation stage, and the magnitude of the compensation optical path is adjusted by moving the translation stage.

[0106] The pulse sequence output by the pulse broadening and amplification module is directed unobstructed toward the dispersion compensation device of the pulse compression module. After being reflected and reduced in height by the second plane mirror, it passes through the dispersion compensation device again and is reflected onto the first plane mirror to output a compressed pulse.

[0107] The spectral broadening and filtering module includes: a mirror, a half-wave plate, a dispersion compensation device, a light-blocking plate, a polarization beam splitter, an aspherical lens, a spectral sidelobe filtering fiber, and an optical filter.

[0108] The pulse sequence output by the pulse broadening and amplification module is directed unobstructed toward the dispersion compensation device, then reflected and reduced in height by the second plane mirror before passing through the dispersion compensation device again, and finally reflected onto the first plane mirror to output a compressed pulse.

[0109] The two pulses emitted from the frequency doubling module and the time delay module are broadened by a transmission grating, collimated by a 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: plane reflector, square reflector, ultraviolet fused silica reflector, microcrystalline glass reflector, silver-plated reflector, gold-plated reflector, preferably selected from one or more of the following: plane reflector, square reflector, silver-plated reflector, more preferably plane reflector or square reflector.

[0111] The self-phase modulation spectral selection driven coherent anti-Stokes Raman microscopy system of this invention can obtain 800nm ​​pump light and 1030nm Stokes light from a 1550nm laser neutron source, realizing coherent anti-Stokes Raman (CARS) microscopic imaging, that is, achieving selective and simultaneous imaging of multiple biological tissue components. Furthermore, the self-phase modulation spectral selection driven microscope system of this invention has strong scalability; the wavelength of the light source can be tuned by adjusting the input fiber energy to suit selective imaging of components in different samples.

[0112] The self-phase-modulated spectrally selected coherent anti-Stokes Raman microscopy system of the present invention may have, but is not limited to, the following beneficial effects:

[0113] 1. The self-phase modulation spectral selection driven coherent anti-Stokes Raman microscope system of the present invention uses the self-phase modulation spectral selection (SESS) method to convert the 1550nm pulse wavelength to 1600nm. Through a frequency doubling crystal, a second harmonic generation process is achieved, converting the 1600nm pulse wavelength to 800nm, thus realizing coherent anti-Stokes Raman microscopy imaging, i.e., selective imaging of various biological tissue components. Compared with other wavelength conversion methods, the device is simple, has high conversion efficiency, and the wavelength is tunable.

[0114] 2. High 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 magnitude of the input fiber energy to suit imaging of different samples. Attached Figure Description

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

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

[0117] Figure labeling: 1. Femtosecond laser front-end module; 2. Dual-wavelength module; 3. Pulse broadening and amplification module; 4. Time delay module; 5. Spectral broadening and filtering module; 6. Second harmonic generation module; 7. Dispersion compensation module; 8. Microscope module; 9. Dual-wavelength fiber isolator; 10. Dual-wavelength pump laser source; 11. Dual-wavelength wavelength division multiplexer; 12. Dual-wavelength fiber amplifier; 13. Highly nonlinear fiber; 14. First fiber broadener; 15. Second fiber broadener; 16. First pulse broadening and amplification pump laser source; 17. Second pulse broadening and amplification pump laser source; 18. First pulse broadening and amplification 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-waveplate; 31. Second pulse-stretching and amplifying half-waveplate; 32. First spatial isolator; 33. 34. Second spatial isolator; 35. First pulse broadening and magnifying mirror; 36. First optical path compensation mirror; 37. Second optical path compensation mirror; 38. Third optical path compensation mirror; 39. Fourth optical path compensation mirror; 40. Fifth optical path compensation mirror; 41. First dispersion compensation half-wave plate; 42. First square mirror; 43. First dispersion compensation transmission grating; 44. Second dispersion compensation transmission grating; 45. First grating mirror; 46. Dispersion compensation mirror; 47. Second pulse broadening and magnifying mirror; 48. First filtering half-wave plate; 49. First filtering mirror; 50. Transmission grating; 51. Second filter transmission grating; 52. Second filter mirror; 53. Second filter half-wave plate; 54. Polarizing beam splitter; 55. Light-blocking plate; 56. Filter focusing lens; 57. Optical fiber; 58. Filter collimating lens; 59. Filter plate; 60. Second harmonic half-wave plate; 61. Second harmonic focusing lens; 62. Second harmonic collimating lens; 63. Second dispersion-compensating half-wave plate; 64. Second square mirror; 65. Third dispersion-compensating transmission grating; 66. Fourth dispersion-compensating transmission grating; 67. Second grating mirror; 68. Dichroic mirror; 69. Microscope.

[0118] A. The ultrashort pulse sequence emitted by femtosecond laser front-end module 1; B. Pulse A is amplified and broadened into wavelengths of 1µm and 1.5µm by nonlinear fiber spectral broadening; C. Pulse B is amplified by chirped pulse amplification module 3 into a dual-wavelength pulse; D. Pulse output by spectral broadening and filtering module 5; E. Pump pulse output by frequency second harmonic generation module 6 for pulse D; F. Pump pulse and Stokes pulse after temporal and spatial overlap. Detailed Implementation

[0119] The present invention will be further described below with reference to the accompanying drawings and specific embodiments. However, it should be understood that these embodiments are merely for more detailed and specific explanation and should not be construed as limiting the present invention in any way.

[0120] Example 1

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

[0122] Figure 1 This 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 spectral broadening and filtering module 5; a second harmonic generation module 6; a dispersion compensation module 7; and a microscope module 8. In this embodiment, the femtosecond laser front-end module 1 employs a ytterbium-doped fiber oscillator with mode-lockable semiconductor-protected absorption mirrors. The parameters corresponding to the output ultrashort pulse sequence A are: center wavelength 1.55 μm and repetition frequency 37 MHz.

[0123] The output ultrashort pulse sequence A is amplified by the dual-wavelength fiber amplifier 12 in the dual-wavelength module 2 and then spectrally broadened by the highly nonlinear fiber 13, resulting in an output spectrum containing pulses B of 1.03µm and 1.55µm. Pulse B is split into two paths and passed through the first fiber broadener 14 and the second fiber broadener 15, respectively. These paths are then amplified by the first pulse broadening and amplification fiber amplifier 20, the second pulse broadening and amplification fiber amplifier 21, the third pulse broadening and amplification fiber amplifier 26, and the fourth pulse broadening and amplification fiber amplifier 27, respectively, to output pulse C, which contains high-power 1.03µm and 1.55µm pulses.

[0124] A pulse with a wavelength of 1.55 μm in pulse C is incident on a grating pair consisting of a first filtering transmission grating 49 and a second filtering transmission grating 50. The first filtering transmission grating 49 and the second filtering transmission grating 50 are placed in parallel, and the second filtering transmission grating 50 is mounted on a precision adjustable displacement platform, which can flexibly control the spacing between the grating pairs. The pulse sequence is refracted downwards by the second filtering reflector 51 and reduced to a certain height before returning and passing through the grating pair. Finally, it is reflected at a 45-degree angle on the first filtering reflector 48 to output a compressed pulse.

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

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

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

[0128] The two pulses of pulse E are respectively adjusted to P-polarization by the first dispersion-compensating half-wave plate 40 and the second dispersion-compensating half-wave plate 63. Then, the second-order dispersion is compensated by the first dispersion-compensating transmission grating 42, the second dispersion-compensating transmission grating 43, the third dispersion-compensating transmission grating 65, and the fourth dispersion-compensating transmission grating 66. Each path of pulse E is broadened to about 3 ps. Afterward, the two light paths are spatially overlapped by the dichroic mirror 68, outputting pulse F, which is then incident on the microscope 69 to image the biological sample.

[0129] Example 2

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

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

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

[0133] A 1550nm pulse is compressed by the first filtering transmission grating 49 and the second filtering transmission grating 50, and then focused into the LEAF fiber 56 by the filtering focusing lens 55. The high-energy narrowband femtosecond pulse can achieve sufficient spectral broadening by propagating within an fiber with a length of 7.5±2.5cm. The broadened spectrum consists of a series of independent spectral sidelobes, with the leftmost and rightmost sidelobes containing a large amount of pulse energy. The light transmitted in the LEAF fiber 56 is collimated by the filtering collimating lens 57. The leftmost / rightmost spectral sidelobes containing the most energy are filtered out by the filter 58, resulting in a near-conversion-limited femtosecond pulse with a wide wavelength tunable, thus achieving wavelength conversion. When the rightmost side of the spectrum broadens to 1600nm, the right sidelobe is filtered out by the filter 58, resulting in the output pulse D.

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

[0135] The 800nm ​​and 1.03µm pulses are compensated for in the dispersion compensation module 7 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, which introduces chirp and increases spectral resolution. Then, spatial alignment is achieved through a dichroic mirror 68 and the pulses are introduced into the microscope 69.

[0136] Using a pulsed F-driven microscope 69, a strong signal can be excited and collected by imaging the CH bonds in a sample using coherent anti-Stokes Raman microscopy. This invention can easily achieve wavelength conversion, obtaining selective imaging suitable for coherent anti-Stokes Raman microscopy.

[0137] Although only one specific embodiment and its effects have been shown above, those skilled in the art should understand that, based on the concept of the present invention, other embodiments not specifically shown or other technical solutions of the present invention not shown in the embodiments can also achieve the same technical effects as those claimed in the summary section:

[0138] 1. The self-phase modulation spectral selection driven coherent anti-Stokes Raman microscope system of the present invention uses the self-phase modulation spectral selection (SESS) method to convert the 1550nm pulse wavelength to 1600nm. Through a frequency doubling crystal, a second harmonic generation process is achieved, converting the 1600nm pulse wavelength to 800nm, thus realizing coherent anti-Stokes Raman microscopy imaging, i.e., selective imaging of various biological tissue components. Compared with other wavelength conversion methods, the device is simple, has high conversion efficiency, and the wavelength is tunable.

[0139] 2. High 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 magnitude of the input fiber energy to suit imaging of different samples.

[0140] Although the invention has been described to a certain extent, it is apparent that appropriate variations can be made to the various conditions without departing from the spirit and scope of the invention. It is understood that the invention is not limited to the described embodiments, but falls within the scope of the claims, which include equivalent substitutions for each of the elements.

Claims

1. A coherent anti-Stokes Raman microscope system, characterized in that, The coherent anti-Stokes Raman microscope system includes: Femtosecond laser front-end module for outputting ultrashort pulse sequences A dual-wavelength module for broadening the spectrum of the ultrashort pulse sequence. A pulse widening and amplification module used to widen the pulse width by introducing dispersion into the widened pulse and then amplify its power. Module used to compensate for the time delay of the optical path difference between two pulses. A spectral broadening and filtering module used to broaden the spectrum of the pulse output from the pulse broadening and amplification module after pulse width compression, and then filter it. A second harmonic generation module for multiplying the pulse output from the spectral broadening and filtering module. A dispersion compensation module for time-domain broadening of dispersion compensation in two-channel pulsed light, and Multiphoton microscope module; Furthermore, the output of the femtosecond laser front-end module is connected to the input of the dual-wavelength module, the output of the dual-wavelength module is connected to the input of the pulse broadening and amplification module, the output of the pulse broadening and amplification module is connected to the input of the time delay module and the spectral broadening and filtering module, the output of the spectral broadening and filtering module is connected to the input of the second harmonic generation module, the outputs of the second harmonic generation module and the time delay module are connected to the input of the dispersion compensation module, and the output of the dispersion compensation module is connected to the input of the multiphoton microscope module. The femtosecond laser front-end module includes an oscillator; wherein, the femtosecond laser front-end module outputs a femtosecond pulse sequence with a repetition frequency of 35~40MHz, and the oscillator in the femtosecond laser front-end module emits an ultrashort pulse sequence with a center wavelength range of 1.50μm~1.66μm, and the oscillator is an erbium-doped fiber laser oscillator; The dual-wavelength module comprises, in sequence: a dual-wavelength fiber isolator, a dual-wavelength pump laser source, a dual-wavelength wavelength division multiplexer, a dual-wavelength fiber amplifier, and a highly nonlinear fiber; The broadened spectrum of the spectral broadening filter module is achieved through a self-phase modulation effect, and / or The multiphoton microscope module is driven by the 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 outputs a femtosecond pulse sequence with a repetition frequency of 37MHz; The oscillator in the femtosecond laser front-end module emits an ultrashort pulse sequence with a center wavelength range of 1.54 μm to 1.57 μm; and / or The oscillator is a mode-locked erbium-doped fiber laser oscillator; The mode-locking method is selected from one or more of the following: semiconductor saturable absorber mirror, nonlinear polarization rotation, and nonlinear optical ring mirror.

3. The coherent anti-Stokes Raman microscope system according to claim 2, characterized in that: The oscillator in the femtosecond laser front-end module emits an ultrashort pulse sequence with a center wavelength range of 1.55 μm; and / or The mode-locking method is a semiconductor saturable absorber mirror.

4. The coherent anti-Stokes Raman microscope system according to any one of claims 1 to 3, characterized in that, The time delay module includes an optical path compensation device; wherein the optical path compensation device includes: an optical path compensation mirror and an optical path compensation translation stage; The optical path compensation mirror is mounted on the optical path compensation translation stage; and / or The number of optical path compensation mirrors is 2 to 10.

5. The coherent anti-Stokes Raman microscope system according to claim 4, characterized in that, The number of optical path compensation mirrors is 4 to 8.

6. The coherent anti-Stokes Raman microscope system according to claim 5, characterized in that, The number of optical path compensation mirrors is 4 or 5.

7. 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 includes a first optical path and a second optical path, wherein the first optical path and the second optical path respectively include: 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. The number of pulse-stretching and magnifying mirrors is 1 to 4; and / or Between the pulse-stretching fiber amplifier and the fiber collimator, the following are also arranged in sequence: 1) The pulse-stretched and amplified pump laser sources in groups 1-3, 2) Fiber optic combiner, and 3) The pulse-stretching and amplifying fiber amplifier.

8. The coherent anti-Stokes Raman microscope system according to claim 7, characterized in that: The number of pulse-stretching and magnifying mirrors is 2 to 3; and / or One to two sets of pulse-stretching amplification pump laser sources are arranged between the pulse-stretching amplification fiber amplifier and the fiber collimator.

9. The coherent anti-Stokes Raman microscope system according to claim 8, characterized in that: The number of pulse-stretching and magnifying mirrors is two; and / or Two sets of pulse-stretching amplification pump laser sources are arranged between the pulse-stretching amplification fiber amplifier and the fiber collimator.

10. The coherent anti-Stokes Raman microscope system according to any one of claims 1 to 3, characterized in that, The spectral broadening and filtering module comprises, in sequence: a first half-wave plate, a pair of filter gratings, a second half-wave plate, a polarization beam splitter, a light-blocking plate, a filter focusing lens, an optical fiber, a filter collimating lens, and a filter plate; The filter grating pair includes a filter mirror pair and a filter transmission grating pair; and / or The optical fiber is selected from one or more of the following: LEAF fiber, photonic crystal fiber, and highly nonlinear fiber.

11. The coherent anti-Stokes Raman microscope system according to claim 10, characterized in that, The optical fiber is either LEAF fiber or photonic crystal fiber.

12. The coherent anti-Stokes Raman microscope system according to claim 11, characterized in that, The optical fiber is a LEAF optical fiber.

13. The coherent anti-Stokes Raman microscope system according to claim 10, characterized in that: The second harmonic generation module sequentially comprises: a second harmonic half-wave plate, a second harmonic focusing lens, a second harmonic crystal, and a second harmonic collimating lens; wherein the second harmonic crystal is selected from one or more of the following: barium metaborate crystal, periodically polarized lithium niobate crystal, lithium iodate; and / or The femtosecond laser front-end module outputs a femtosecond pulse sequence, which is then divided into a first optical path and a second optical path after passing through the dual-wavelength module and the pulse broadening and amplification module. The pulses output from the first optical path enter the spectral broadening and filtering module, are adjusted to P-polarization by the first half-wave plate, and are then focused onto the optical fiber by the filtering and focusing lens for spectral broadening. After passing through the filtering and collimating lens, the spectrally broadened pulses are collimated and output, and then enter the second harmonic generation module. The pulses output from the second optical path enter the time delay module.

14. The coherent anti-Stokes Raman microscope system according to claim 13, characterized in that, The frequency doubling crystal is a barium borate crystal or a periodically polarized lithium niobate crystal.

15. The coherent anti-Stokes Raman microscope system according to claim 14, characterized in that, The frequency doubling crystal is a periodically polarized lithium niobate crystal.

16. The coherent anti-Stokes Raman microscope system according to any one of claims 1 to 3, characterized in that, The dispersion compensation module includes: a dispersion compensation device group, a dispersion compensation mirror, and a dichroic mirror; wherein: The dispersion compensation device group includes: a dispersion compensation grating pair, a grating mirror 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 dispersion compensation device groups is at least 2; The two pulses output by the frequency harmonic generation module and the time delay module are broadened by the dispersion compensation module and then spatially overlapped by the dispersion compensation module before being imported into the multiphoton microscope module.

17. The coherent anti-Stokes Raman microscope system according to claim 16, characterized in that, The two pulses output by the frequency harmonic generation module and the time delay module are broadened by the dispersion compensation module and then spatially overlapped by the dichroic mirror before being introduced into the multiphoton microscope module.

18. The coherent anti-Stokes Raman microscope system according to claim 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 selected from one or more of the following: plane reflector, square reflector, ultraviolet fused silica reflector, microcrystalline glass reflector, silver-plated reflector, gold-plated reflector. The dual-wavelength pump laser source and / or the pulse-stretching amplification pump laser source are each 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; and / or The dual-wavelength fiber amplifier and / or pulse-stretching fiber amplifier are each selected from one or more of the following: Yb-doped single-mode fiber amplifier, Yb-doped multimode fiber amplifier, Er-doped single-mode fiber amplifier, Er-doped multimode fiber amplifier, and Yb / Er co-doped multimode fiber amplifier; wherein: The Yb-doped single-mode fiber amplifier is a Yb401 single-mode fiber amplifier. The Yb-doped multimode fiber amplifier is a Yb12 / 125 multimode fiber amplifier and / or a Yb30 / 250 multimode fiber amplifier. The Er-doped single-mode fiber amplifier is an Er80 single-mode fiber amplifier and / or an Er40 single-mode fiber amplifier; and / or The Yb / Er co-doped multimode fiber amplifier is a Yb / Er30 / 250 multimode fiber amplifier.

19. The coherent anti-Stokes Raman microscope system according to claim 18, characterized in that: The mirrors in the optical path compensation mirror, pulse broadening and magnification mirror, dispersion compensation mirror, grating mirror pair, and / or filter mirror pair are all selected from one or more of the following: plane mirror, square mirror, silver-plated mirror; and / or The dual-wavelength pumped laser source and / or the pulse broadening and amplification pumped laser source are both 1W diode pumped laser sources or 27W diode pumped laser sources.

20. The coherent anti-Stokes Raman microscope system according to claim 19, characterized in that, The mirrors in the optical path compensation mirror, pulse broadening and magnification mirror, dispersion compensation mirror, grating mirror pair and / or filter mirror pair are all planar mirrors or square mirrors.

21. A method for driving a microscope, characterized in that, The method includes: driving the microscope using the coherent anti-Stokes Raman microscope system according to any one of claims 1 to 20; The method includes: the femtosecond laser front end emits an ultrashort pulse sequence, which is then spectrally broadened by the dual-wavelength module, and then outputs two power-amplified ultrashort pulse sequences by the pulse broadening and amplification module. One of the power-amplified ultrashort pulse sequences is time-delayed by the time delay module, and the other is wavelength-adjusted by the spectral broadening and filtering module. The wavelength is then converted by the frequency second harmonic generation module, and the pulses output by the time delay module are input together with the pulses to the dispersion compensation module for broadening. Finally, the pulses are collimated and input into the multiphoton microscope module to achieve sample imaging.

22. A nonlinear optical microscope device, characterized in that: The nonlinear optical microscopy device includes the coherent anti-Stokes Raman microscope system according to any one of claims 1 to 20, wherein the nonlinear optical microscopy device is a microscope driven by self-phase modulation spectral selection.

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