High signal-duty-cycle dual optical frequency comb coherent anti-Stokes Raman imaging system

Abstract

This invention provides a high signal duty cycle dual-optical frequency comb coherent anti-Stokes Raman imaging system, belonging to the field of coherent anti-Stokes Raman imaging technology. It solves the problems of low signal duty cycle and low measurement speed of traditional dual-optical frequency comb output pulses. The system includes a tunable repetition rate difference dual-optical frequency comb pulse generation module, an optical focusing and sample scanning module, and a data acquisition module. The tunable repetition rate difference dual-optical frequency comb pulse generation module generates two dual-optical frequency comb pulse beams, one with an adjustable repetition frequency and the other with a fixed repetition frequency. The pulse beam with the adjustable repetition frequency serves as the pump light, while the pulse beam with the fixed repetition frequency utilizes the nonlinear effect of highly nonlinear optical fibers to achieve spectral broadening and spectral frequency shift, obtaining Stokes light. This invention is applied to anti-Stokes Raman imaging.

Description

Technical Field

[0001] The invention provides a dual optical frequency comb coherent anti-Stokes Raman imaging system with a high signal duty cycle, belonging to the technical field of coherent anti-Stokes Raman imaging. Background Art

[0002] High coherence anti-Stokes Raman imaging is a third-order nonlinear optical process based on the four-wave mixing effect. It has a wide range of applications in biological sample analysis, cancer cell identification, and pharmacokinetics due to its label-free and non-invasive characteristics. Compared with traditional optical microscopy, coherent anti-Stokes Raman imaging can provide rich chemical component information in samples, thereby helping scientists understand the changes in various material components during life activities. To achieve coherent anti-Stokes Raman imaging, two ultrashort pulses are usually used as pump light and probe light, respectively, and irradiated onto the sample to be tested at the same time. When the energy corresponding to the frequency difference of the two beams of light is consistent with the vibrational and rotational energy levels of the sample's chemical bonds, an anti-Stokes photon will be generated. By detecting its intensity and position, the detection of material molecules can be achieved. Usually, coherent anti-Stokes Raman imaging technology relies on a single laser, combined with mechanical delayed scanning to delay sampling of pulses, but the stability of mechanical scanning and the long delay scanning time greatly limit the sampling speed and stability.

[0003] In recent years, some researchers have proposed the application of dual optical frequency comb technology in coherent anti-Stoke Raman imaging, but the light sources used are mostly two sets of lasers. Since a precise feedback control system is required to eliminate the randomness of the output frequencies of the two sets of lasers, the technical difficulty and high price of the technology limit the use of coherent anti-Stoke Raman imaging outside the laboratory. In addition, the output pulse repetition frequency and repetition frequency difference of the traditional dual optical frequency comb are fixed, and the ratio of the signal duration to the automatic delay scanning time is very small, resulting in a serious decrease in the signal duty cycle and measurement speed, resulting in a waste of measurement time and a reduction in the signal-to-noise ratio. Summary of the invention

[0004] In order to solve the above technical problems, the present invention proposes a dual optical frequency comb coherent anti-Stokes Raman imaging system with a high signal duty cycle, which can significantly improve the dual optical frequency comb signal duty cycle and sampling speed, and realize rapid label-free imaging of samples.

[0005] The technical solution adopted by the present invention is: a high signal duty cycle dual optical frequency comb coherent anti-Stokes Raman imaging system, comprising a dual optical comb pulse generation module with tunable repetition frequency difference, an optical focusing and scanning sample module and a data acquisition module, wherein the dual optical comb pulse generation module with tunable repetition frequency difference is used to generate two beams of dual optical frequency comb pulse light, wherein the repetition frequency of one beam of dual optical frequency comb pulse light is adjustable, and the repetition frequency of the other beam of dual optical frequency comb pulse light is fixed, the pulse light with adjustable repetition frequency is used as pump light, and the pulse light with fixed repetition frequency utilizes the nonlinear effect of high nonlinear optical fiber to achieve spectral broadening and spectral frequency shift to obtain Stokes light;

[0006] The pump light and Stokes light are simultaneously incident on the sample in the optical focusing and scanning sample module to jointly excite the molecules to generate anti-Stokes light, and the molecular information at different positions is scanned by moving the sample;

[0007] The data acquisition module collects anti-Stokes optical signals.

[0008] The repetition frequency difference tunable dual optical comb pulse generation module includes a pump source, a fiber laser and a transmission optical path. The pump source is used to generate an excitation light source, and the excitation light source is injected into the erbium-doped optical fiber that provides a gain medium for the laser through a wavelength division multiplexer. The fiber laser is provided with two sets of optical paths for clockwise and counterclockwise propagation.

[0009] Two circulators are used in the fiber laser to control the light beam to transmit in the clockwise and counterclockwise directions respectively; in the clockwise branch, a single-walled carbon nanotube saturable absorber is used in combination with a polarization controller for pulse mode locking, and an electrically controlled polarization controller and a birefringent crystal are introduced. By periodically adjusting the voltage loaded on the polarization controller, the polarization state of the incident birefringent crystal is adjusted, thereby periodically changing the phase delay and dispersion of the pulse light propagation, resulting in a change in the repetition frequency of the output optical frequency comb; in the counterclockwise branch, a single-walled carbon nanotube saturable absorber is used in combination with a polarization controller for pulse mode locking;

[0010] The laser light generated by the fiber laser is divided into two parts by an optical coupler, one part of the laser light continues to be transmitted in the fiber laser cavity, and the other part outputs counterclockwise and clockwise optical frequency combs for unidirectional propagation through polarization-independent isolators respectively. The counterclockwise optical frequency comb is used to generate 1550nm laser light as pump light through an erbium-doped fiber amplifier, and the clockwise optical frequency comb is used to generate 1060nm laser light as Stokes light through erbium-doped fiber amplifier, high nonlinear fiber and ytterbium-doped fiber amplifier for spectral broadening.

[0011] The power is adjusted in the counterclockwise branch through an adjustable attenuator.

[0012] In the optical focusing and scanning sample module, the pump light passes through a collimator and a reflector and is combined with the Stokes light that passes through the collimator in a dichroic mirror. The two light frequency combs are focused on the sample through a low-pass filter and a focusing objective lens to jointly excite the molecules to produce anti-Stokes light. The light is then filtered out through a short-pass filter to remove the original excitation signal before being incident on the data acquisition module.

[0013] Avalanche photodiodes are used in the data acquisition module for photoelectric conversion and signal detection.

[0014] In the optical focusing and scanning sample module, two pulsed light beams coherently excite the molecules so that the Raman shift wavenumber range matches that of the sample to be measured.

[0015] The spectral range of the pump light is between 10-20 nm with a central wavelength of 1550 nm, and its repetition frequency is 50 MHz.

[0016] The Raman shift measured by the avalanche photodiode is:

[0017] ;

[0018] Where: K is the Raman shift, c is the speed of light, l pump is the pump light wavelength, l stocks is the Stokes wavelength, Δ oh is the frequency difference, Δ ω=ω pump - oh stocks , oh pump is the frequency of the pump light, oh stocks is the frequency of Stokes light.

[0019] The beneficial effects of the present invention compared with the prior art are as follows: the coherent anti-Stokes Raman imaging implemented by the system of the present invention has the following advantages:

[0020] 1. The laser proposed in the present invention uses an electrically controlled polarization controller to adjust the polarization state of the pulse injected into the birefringent crystal, change the phase delay and dispersion of the pulse light propagation, and accurately adjust the repetition frequency of the pulse light output;

[0021] 2. The laser proposed in the present invention improves the duty cycle through high repetition frequency, shortens the sample acquisition time, and provides a new method for coherent anti-Stokes Raman imaging;

[0022] 3. The present invention is based on the coherent Raman principle and performs label-free imaging of samples without causing damage to biological samples;

[0023] 4. The present invention has no requirement on the wavelength of incident light based on the coherent Raman principle, and infrared light can be used as the light source. For biological samples, longer wavelengths can achieve greater penetration depth due to reduced scattering within the sample. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] The present invention will be further described below in conjunction with the accompanying drawings:

[0025] Figure 1 It is a schematic diagram of the system structure of the present invention;

[0026] Figure 2 This is the principle diagram of the traditional dual optical frequency comb coherent Raman imaging signal acquisition;

[0027] Figure 3 This is a schematic diagram of the signal acquisition principle of coherent anti-Stokes Raman imaging with high signal duty cycle;

[0028] In the figure: 101: repetition frequency difference tunable dual optical comb pulse generation module, 102: optical focusing and scanning sample module, 103: data acquisition module;

[0029] Pump Laser: pump source, WDM: wavelength division multiplexer, EDF: erbium-doped fiber, ISO: polarization-independent isolator, PC: polarization controller, OC: coupler, SWNT: single-walled carbon nanotube saturable absorber, SMF: single-mode fiber, OA: tunable attenuator, CIR: circulator, DC: birefringent crystal, EDFA: erbium-doped fiber amplifier, YDFA: ytterbium-doped fiber amplifier, HNLF: highly nonlinear fiber, COL: collimator, M: reflector, DM: dichroic mirror, LPF: low-pass filter, OB: focusing objective, Sample: sample, SPF: short-pass filter, APD: avalanche photodiode. DETAILED DESCRIPTION

[0030] like Figure 1 to Figure 3As shown, the present invention provides a dual optical frequency comb coherent anti-Stokes Raman imaging system with a high signal duty cycle, comprising a dual optical comb pulse generation module 101 with tunable repetition frequency difference, an optical focusing and scanning sample module 102 and a data acquisition module 103, wherein the dual optical frequency comb pulse generation module 101 with tunable repetition frequency difference comprises a dual optical frequency comb laser, which is internally divided into two branches, one of which adjusts the repetition frequency of the pulse output by adjusting the polarization state of the incident birefringent crystal to change the phase delay and dispersion of the pulse light propagation, and the repetition frequency of the mode-locked pulse output by the other branch is a fixed value, thereby obtaining two beams of dual optical frequency comb pulse lights with different repetition frequencies. The pulse light with adjustable repetition frequency is used as pump light by generating 1550nm band laser through erbium-doped fiber amplifier. The spectral range of pump light is about 10-20nm, and its repetition frequency is about 50MHz. The pulse light with fixed repetition frequency first passes through erbium-doped fiber amplifier, then spectrally broadens through high nonlinear fiber, and then is injected into ytterbium-doped fiber amplifier to obtain 1060nm band laser as Stokes light. In the optical focusing and scanning sample module 102, the pump light and Stokes light are incident on the sample to be tested at the same time, jointly excite the molecules to generate anti-Stokes light, and scan the molecular information at different positions by moving the sample. The data acquisition module 103 collects anti-Stokes light signals through photoelectric conversion by avalanche photodiode.

[0031] Specifically, the repetition frequency difference tunable dual optical comb pulse generation module 101 includes an optical fiber laser and an optical transmission path that can generate two sets of optical frequency combs, and the two sets of optical frequency combs can propagate in a clockwise (CW) and counterclockwise (CCW) direction, respectively. A 980nm pump source (Pump Laser) is used to provide an excitation light source for the present invention, and the pump source uses a semiconductor laser tube. The excitation light source is injected into the erbium-doped fiber (EDF) that provides a gain medium for the optical fiber laser through a wavelength division multiplexer (WDM). Two circulators (CIR) control the light beam to be transmitted along CW and CCW, respectively. On the CW branch, a single-walled carbon nanotube saturable absorber (SWNT2) is combined with a polarization controller (PC) for pulse mode locking. At the same time, an electrically controlled polarization controller (PC) and a birefringent crystal (DC) are introduced, and the polarization state of the incident birefringent crystal is adjusted by periodically adjusting the voltage loaded on the polarization controller, thereby periodically changing the phase delay and dispersion size of the pulse light propagation, resulting in a change in the repetition frequency of the output optical frequency comb. In the CCW branch, a single-walled carbon nanotube saturable absorber (SWNT1) is also introduced in combination with a polarization controller (PC) for pulse mode locking. The repetition frequency of this output is a fixed value. In order to balance the power difference between CW and CCW, a tunable attenuator (OA) is introduced to adjust the power. The 5:5 optical coupler (OC) divides the laser generated by the fiber laser into two parts, 50% of which is used as output, and the other 50% of the laser continues to be transmitted in the fiber laser cavity. After the CCW and CW optical frequency combs output by the fiber laser pass through polarization-independent isolators (ISO1 and ISO2) to ensure two-way propagation, one of them is used to generate 1550nm band laser through an erbium-doped fiber amplifier (EDFA1), and the other is used to generate 1060nm band laser through erbium-doped fiber amplifier (EDFA2), high nonlinear fiber (HNLF) and ytterbium-doped fiber amplifier (YDFA) for spectral broadening. One beam is used as pump light (pump) and the other is used as Stokes light (stocks).

[0032] The repetition frequency difference between the two output pulse lights can be precisely tuned by adjusting the polarization state, and the output pulse power after the amplifier is about 100 milliwatts.

[0033] Then, in the optical focusing and scanning sample module 102, the pump light passes through the collimator (COL1) and the reflector (M), and the Stokes light passes through the collimator (COL2). The two beams of light are combined in the dichroic mirror (DM), and the two light frequency combs are focused on the sample (Sample) through the low-pass filter (LPF) and the focusing objective lens (OB1 and OB2), and jointly excite the molecules to generate anti-Stokes light (anti-stocks). After the original excitation signal is filtered out by the short-pass filter (SPF), it is incident on the avalanche photodiode (APD) of the data acquisition module 103 for photoelectric conversion and signal detection.

[0034] In the optical focusing and scanning sample module 102, the Raman shift wave number range of the molecules coherently excited by the two pulsed lights matches that of the sample to be measured.

[0035] The relationship between the fundamental frequency mode-locked pulse repetition frequency and the laser cavity length of the pulsed light in the 1550nm band is as follows:

[0036] f 1= c / nL 1;

[0037] The relationship between the fundamental frequency mode-locked pulse repetition frequency and the laser cavity length of the pulsed light in the 1060nm band is as follows:

[0038] f 2= c / ( nL 2+Δ nl );

[0039] Where: f 1 and f 2 are the fundamental repetition frequencies of 1550nm and 1060nm pulsed light, L 1 and L 2 are the cavity lengths of the 1550nm and 1060nm lasers, respectively. c is the speed of light, n is the refractive index of the optical fiber, l is the thickness of the birefringent crystal, Δ n is the refractive index change caused by the dispersion change in the crystal. n Periodic changes make f 1 and f The difference of 2 changes periodically, so that the two pulsed lights can act throughout the whole process.

[0040] The delay calculation formula of the dual optical frequency comb group is as follows:

[0041] Δ t =1 / f 1-1 / f 2.

[0042] like Figure 2 As shown in Figure 1, the conventional dual optical frequency comb coherent Raman imaging has a long time window period and a very low duty cycle, where the duty cycle of the conventional dual optical frequency comb is defined as:

[0043] DC =Δ f · t / Δ t · f 1;

[0044] Refresh time:

[0045] T refresh =1 / Δ f ;

[0046] Where: Δ f is the repetition frequency difference, t is the signal duration.

[0047] like Figure 3 As shown, the high-signal and high-duty-cycle dual optical frequency comb coherent anti-Stokes Raman imaging acquisition system proposed by the present invention has a periodic change in the intracavity repetition frequency caused by periodic adjustment of the polarization state, so the basic window period is significantly reduced, and the duty cycle can almost reach a very high level, and its refresh time is:

[0048] T refresh = t / Δ t · f 1.

[0049] The Raman shift measured in the data acquisition module 103 can be obtained by the following formula:

[0050] oh anti-stocks =2 oh pump - oh stocks ;

[0051] Δ ω=ω pump - oh stocks ;

[0052] ;

[0053] Where: oh anti-stocks is the frequency of the anti-Stokes light, oh pump is the frequency of the pump light, oh stocks is the frequency of Stokes light, Δ oh is the frequency difference, K is the Raman shift, c is the speed of light, l pump is the pump light wavelength, l stocks is the Stokes wavelength;

[0054] The wavelength is calculated as follows:

[0055] ;

[0056] ;

[0057] Where: c is the wave number, l is the wavelength, oh is the frequency.

[0058] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A high signal duty cycle dual optical frequency comb coherent anti-Stokes Raman imaging system, characterized by: It comprises a dual optical comb pulse generation module with tunable repetition frequency difference, an optical focusing and scanning sample module and a data acquisition module. The dual optical comb pulse generation module with tunable repetition frequency difference is used to generate two beams of dual optical frequency comb pulse light, wherein the repetition frequency of one beam of dual optical frequency comb pulse light is adjustable, and the repetition frequency of the other beam of dual optical frequency comb pulse light is fixed. The pulse light with adjustable repetition frequency is used as pump light, and the pulse light with fixed repetition frequency uses the nonlinear effect of high nonlinear optical fiber to achieve spectrum broadening and spectrum frequency shift to obtain Stokes light. The pump light and Stokes light are simultaneously incident on the sample in the optical focusing and scanning sample module to jointly excite the molecules to generate anti-Stokes light, and the molecular information at different positions is scanned by moving the sample; The data acquisition module collects anti-Stokes optical signals; The repetition frequency difference tunable dual optical comb pulse generation module includes a pump source, a fiber laser and a transmission optical path. The pump source is used to generate an excitation light source, and the excitation light source is injected into the erbium-doped optical fiber that provides a gain medium for the laser through a wavelength division multiplexer. The fiber laser is provided with two sets of optical paths for clockwise and counterclockwise propagation. Two circulators are used in the fiber laser to control the light beam to transmit in the clockwise and counterclockwise directions respectively; in the clockwise branch, a single-walled carbon nanotube saturable absorber is used in combination with a polarization controller for pulse mode locking, and an electrically controlled polarization controller and a birefringent crystal are introduced. By periodically adjusting the voltage loaded on the polarization controller, the polarization state of the incident birefringent crystal is adjusted, thereby periodically changing the phase delay and dispersion of the pulse light propagation, resulting in a change in the repetition frequency of the output optical frequency comb; in the counterclockwise branch, a single-walled carbon nanotube saturable absorber is used in combination with a polarization controller for pulse mode locking; The laser light generated by the fiber laser is divided into two parts by an optical coupler, one part of the laser light continues to be transmitted in the fiber laser cavity, and the other part outputs counterclockwise and clockwise optical frequency combs for unidirectional propagation through polarization-independent isolators respectively. The counterclockwise optical frequency comb is used to generate 1550nm laser light as pump light through an erbium-doped fiber amplifier, and the clockwise optical frequency comb is used to generate 1060nm laser light as Stokes light through erbium-doped fiber amplifier, high nonlinear fiber and ytterbium-doped fiber amplifier for spectral broadening.

2. The high signal duty cycle dual optical frequency comb coherent anti-Stokes Raman imaging system according to claim 1, characterized in that: The power is adjusted in the counterclockwise branch through an adjustable attenuator.

3. The high signal duty cycle dual optical frequency comb coherent anti-Stokes Raman imaging system according to claim 1, characterized in that: In the optical focusing and scanning sample module, the pump light passes through a collimator and a reflector and is combined with the Stokes light that passes through the collimator in a dichroic mirror. The two light frequency combs are focused on the sample through a low-pass filter and a focusing objective lens to jointly excite the molecules to produce anti-Stokes light. The light is then filtered out through a short-pass filter to remove the original excitation signal before being incident on the data acquisition module.

4. The high signal duty cycle dual optical frequency comb coherent anti-Stokes Raman imaging system according to claim 3, characterized in that: Avalanche photodiodes are used in the data acquisition module for photoelectric conversion and signal detection.

5. The high signal duty cycle dual optical frequency comb coherent anti-Stokes Raman imaging system according to claim 4, characterized in that: In the optical focusing and scanning sample module, two pulsed light beams coherently excite the molecules so that the Raman shift wavenumber range matches that of the sample to be measured.

6. The high signal duty cycle dual optical frequency comb coherent anti-Stokes Raman imaging system according to claim 1, characterized in that: The spectral range of the pump light is between 10-20 nm with a central wavelength of 1550 nm, and its repetition frequency is 50 MHz.

7. The high signal duty cycle dual optical frequency comb coherent anti-Stokes Raman imaging system according to claim 5, characterized in that: The Raman shift measured by the avalanche photodiode is: ； Where: K is the Raman shift, c is the speed of light, λ pump is the pump light wavelength, λ stocks is the Stokes wavelength, Δ ω is the frequency difference, Δ ω=ω pump -ω stocks , ω pump is the frequency of the pump light, ω stocks is the frequency of Stokes light.

Images