Miniature high-temporal-spatial-resolution Raman microscope, construction method and application

Abstract

The invention discloses a miniature Raman microscope with high spatial-temporal resolution. The miniature Raman microscope comprises a light source part, a micro-electro-mechanical scanning subsystem, a high-speed signal acquisition and storage subsystem and a drive control and data processing subsystem. The light source part adopts a double-optical-comb pulse light source; the micro-electro-mechanical scanning subsystem comprises a multimode optical fiber, a micro-electro-mechanical system and a gradient refractive index lens; the high-speed signal acquisition and storage subsystem comprises a short-pass filter LFP, a high-speed photoelectric detector APD and a high-speed data acquisition card; and the driving control and data processing subsystem comprises a driving control module and a data processing module and is used for controlling the light source part, the micro-electro-mechanical scanning subsystem and the high-speed signal acquisition and storage subsystem and processing the obtained data. The invention further provides a building method and application of the miniature high-temporal-spatial-resolution Raman microscope.

Description

Technical Field

[0001] The present invention belongs to the field of spectral imaging and biosensor technology, and relates to a miniature high-temporal and spatial resolution Raman microscope and a construction method and application thereof. Background Art

[0002] As the most advanced part of the central nervous system, changes in the internal environment of the brain can cause a series of diseases. Especially in the deep brain region, changes in its internal environment have more important pathological significance. Conducting research related to the deep brain region is conducive to clarifying the structure and function of the nervous system, thereby revealing the neural mechanism of brain work and providing a more reliable basis for the diagnosis and treatment of related diseases. Traditional functional magnetic resonance imaging and electrophysiological techniques are widely used in brain imaging analysis, but they are generally limited by low spatial resolution and it is difficult to distinguish the chemical signals of different substances. Raman spectroscopy can provide fingerprint information of molecules, has high spectral resolution and the characteristics of resistance to photobleaching and autofluorescence, and is very suitable for biological imaging. However, traditional Raman imaging devices are still difficult to use for in vivo microscopic imaging, especially in deep brain regions. This is mainly because its imaging method is based on the microscopic Raman imaging technology of the grating spectrometer, which makes the imaging time very long, generally taking several hours. In addition, the Raman instrument based on the desktop microscope is large in size and cannot be used for freely moving living animals. Therefore, there is an urgent need to develop a miniature high-temporal and spatial resolution Raman microscope to achieve high-throughput real-time analysis and imaging of neurochemical substances in deep brain regions. Summary of the invention

[0003] In order to solve the problem of poor temporal and spatial resolution of imaging in the prior art, the purpose of the present invention is to provide a method for building a miniature high temporal and spatial resolution Raman microscope and its application, construct a dual-comb light source, innovatively integrate multimode optical fiber, micro-electromechanical, and gradient refractive index microlenses, and combine high-speed photodetectors and high-speed data acquisition cards to develop a miniature fiber Raman imaging system with high temporal and spatial resolution and high spectral resolution. The miniature high temporal and spatial resolution Raman microscope has high temporal and spatial resolution and can realize high-resolution rapid acquisition and / or imaging of Raman signals of cells and different brain regions.

[0004] The use of micro-electromechanical systems in the present invention simplifies the structure of the entire microscope, improves the problem of large volume of Raman instruments in existing microscopes, and realizes miniaturization of the instrument.

[0005] The present invention provides a miniature Raman microscope with high temporal and spatial resolution, as shown in the attached Figure 4 shown.

[0006] The micro Raman microscope with high temporal and spatial resolution comprises the following components:

[0007] (1) Light source part (dual-comb pulse light source). The core technology of the light source part is a dual-comb pulse light source with different frequencies; two light combs with slightly different repetition frequencies are used as light sources in the dual-comb spectrum, and the frequencies are f1 and f2 respectively, and the repetition frequency difference of the dual-comb beat signal is Δf = f2-f1. Specifically, the dual-comb pulse light source part includes a fixed-repetition-frequency light comb pulse light source for generating ultrashort pulses, a chirped pulse amplification technology for a large-mode-field double-clad optical fiber for amplifying high-power lasers, and a repetition frequency locking technology for precisely controlling the light field, a carrier-envelope phase control technology, and a feedforward light comb control technology for an acousto-optic frequency shifter. The light source part also includes a collimator (a first collimator and a second collimator) and a lens group (a first reflector and a first dichroic mirror) for adjusting and transmitting the optical path of the excitation light.

[0008] (2) Micro-electromechanical scanning subsystem. This part mainly includes multi-mode optical fiber, micro-electromechanical system and gradient refractive index lens. The multi-mode optical fiber adopts a multi-mode optical fiber with a central wavelength of 1064nm and a mode field diameter of 6.7μm, which is used for the transmission of excitation light and the synchronous collection of Raman signals, and the pulse broadening and dispersion caused by the nonlinear effect of the optical comb light source are suppressed to the maximum extent. The working wavelength loss is <100dB / km, and the dispersion is <1ps / nm.km. While ensuring the efficient excitation of Raman signals, the impact on the free movement of animals is minimized. The nonlinear effect of the light source refers to the exchange of energy when the light wave and the medium interact, which reduces the energy of the photons and changes the light frequency, thereby causing the dispersion and broadening of the pulse. The excitation light is transmitted through the optical fiber and collimated before entering the MEMS. After being reflected by the MEMS, the excitation light enters the gradient refractive index lens. According to the actual application needs, a large numerical aperture (NA=0.8) gradient refractive index lens (GRIN) with different lengths (1-5mm) and diameters (0.5-2.0mm) is used. The GRIN lens focuses the excitation light onto the scanning plane to achieve high spatial resolution confocal scanning under the electrical drive signal, as shown in the attached figure. Figure 5 shown.

[0009] (3) High-speed signal acquisition and storage subsystem. This subsystem mainly includes a short-pass filter (LFP), a high-speed silicon photodetector (APD), and a high-speed data acquisition card (Digitizer). In order to improve the detection signal-to-noise ratio, the device uses a short-pass filter to filter the excitation light in the detection signal. The high-speed silicon photodetector with variable gain and integrated thermistor converts the optical signal into an electrical signal in real time, which is then read and stored in real time by the high-speed data acquisition card, as shown in the attached figure. Figure 6 shown.

[0010] (4) Instrument drive control and data processing subsystem, which includes a drive control module and a data processing module, and is used to control the light source part, the micro-electromechanical scanning subsystem, the high-speed signal acquisition and storage subsystem, and process the acquired data. Specifically, it supports the output frequency and output power adjustment of the dual-comb light source, controls the micro-electromechanical (MEMS) fast confocal scanning, the real-time signal acquisition of the high-speed photodetector and the real-time data storage of the high-speed data acquisition card, and provides a development window. This part uses the interaction between the dual-comb light source and the probe to generate a series of beat frequency signals, and performs Fourier transform (FFT) to obtain Raman spectral data. Fourier transform data processing is based on the following formula.

[0011] F(ω)=∫f(t)·e -iωt dt

[0012] When f(t) is a non-periodic continuous function, where F(ω) represents the frequency domain signal, f(t) represents the time domain signal, ω is the angular frequency, t is the time, and e^(-iωt) is a complex exponential function.

[0013] The general process of data processing is as follows Figure 7 shown.

[0014] In addition to the above-mentioned dual-comb pulse light source, micro-electromechanical scanning subsystem, high-speed signal acquisition subsystem, drive control and data processing subsystem, it also includes an additional second reflector, a second dichroic mirror, a third collimator, and a multi-mode optical fiber; which are used to adjust the propagation path of the laser, collimate the laser, and transmit the laser.

[0015] The present invention provides a method for constructing a miniature high-temporal-spatial resolution Raman microscope, which requires constructing a hardware structure including a light source part, a micro-electromechanical scanning subsystem, a high-speed signal acquisition and storage subsystem, and a drive control and data processing subsystem, and also requires debugging the constructed hardware structure, and the debugging includes the following steps:

[0016] Step 1: Generation and tuning of the optical comb, including the following sub-steps:

[0017] Step 1-1: Use fiber femtosecond laser as stable pump to generate coherent excitation;

[0018] Step 1-2: Tuning is performed through the spatial optical path, so that a dual-comb light source with a fixed frequency difference Δf, adjustable intensity and spot size is finally obtained after tuning the spatial optical path;

[0019] Step 2: MEMS scanning subsystem integration and debugging, including the following sub-steps:

[0020] Step 2-1: Based on the sensitivity of the deformation of the piezoelectric ceramic to the applied voltage, the angle of the reflector in the micro-electromechanical system (MEMS) is changed by changing the applied voltage, thereby realizing the control of the scanning optical path;

[0021] Step 2-2: Use a high numerical aperture gradient refractive index (GRIN) lens to focus the excitation light onto the scanning plane to achieve high-resolution imaging under an electrical drive signal;

[0022] Step 2-3: Use multimode optical fiber as the transmission medium of pulsed laser to reduce the nonlinear broadening and dispersion of the excitation light and ensure the excitation efficiency;

[0023] Step 3: Light path adjustment and calibration, including the following sub-steps:

[0024] Step 3-1: Use two optical combs with slightly different repetition frequencies (Δf) as pulse light sources. After passing through the second dichroic mirror, they are focused by the collimator and enter the multimode optical fiber, and then enter the micro-electromechanical scanning subsystem at the other end of the optical fiber. Δf = f2-f1, where f1 and f2 are the frequencies of the two optical combs respectively.

[0025] Step 3-2: The excitation light and the detection light are located at the same end of the optical fiber, the micro-electromechanical scanning subsystem is located at the other end of the optical fiber, and both ends of the optical fiber are fixed on the optical platform to ensure the effective transmission of the optical signal.

[0026] The present invention also provides the use of a micro Raman microscope with high temporal and spatial resolution constructed by the method described above in in vitro and / or in vivo Raman signal collection and / or imaging.

[0027] The present invention also provides the use of a high spatiotemporal resolution micro-Raman microscope constructed by the method as described above in collecting in vitro Raman molecular signals under excitation light, wherein the Raman molecules include rhodamine B, p-mercaptobenzoic acid, and cyanine dye 5.

[0028] The excitation light wavelength is selected from 633 nm (point value) and 785 nm (point value), preferably 785 nm.

[0029] Wherein, the concentration of the Raman molecules is 0.1-5 mM, preferably 1 mM.

[0030] Among them, the scanning range is 100-3200cm -1 , preferably, 1000-1800cm -1 .

[0031] The present invention also provides the use of a micro Raman microscope with high temporal and spatial resolution constructed by the method as described above in the collection and / or imaging of Raman molecular signals in cells under excitation by excitation light.

[0032] Wherein, the wavelength of the excitation light is selected from one of 633nm (point value) and 785nm (point value), preferably, 785nm.

[0033] Wherein, the Raman molecule includes rhodamine B, p-mercaptobenzoic acid, and cyanine dye 5; preferably, p-mercaptobenzoic acid.

[0034] Among them, the scanning range is 100-3200cm -1 , preferably, 1000-1800cm -1 .

[0035] The present invention also provides the use of a high spatiotemporal resolution micro-Raman microscope constructed by the method described above in collecting and / or imaging Raman molecular signals in different brain regions under excitation light, wherein the different brain regions include the cortex, hippocampus, and thalamus.

[0036] Wherein, the wavelength of the excitation light is selected from one of 633nm (point value) and 785nm (point value), preferably, 785nm.

[0037] Wherein, the Raman molecule includes rhodamine B, p-mercaptobenzoic acid, and cyanine dye 5; preferably, p-mercaptobenzoic acid.

[0038] Among them, the scanning range is 100-3200cm -1 , preferably, 1000-1800cm -1 .

[0039] The beneficial effects of the present invention are as follows: the present invention constructs a micro Raman microscope with high temporal and spatial resolution, and a micro scanning microscope based on microelectromechanical system (MEMS) scanning and a high numerical aperture value gradient refractive index (GRIN) lens realizes three-dimensional Raman scanning in vivo, and realizes an imaging spatial resolution of 200-400nm; the advantages of high spectrum stability, wide band coherence, and time domain precision control performance of the dual light comb light source are combined to increase the Raman imaging speed by 1000 times to the second level; the spectral resolution is increased by 100 times to 0.001nm; the collection photonic crystal fiber is used as the excitation and collection fiber to improve the Raman signal excitation efficiency; in addition, a high-speed and high-sensitivity photodetector is used to collect signals in real time to further improve the imaging signal-to-noise ratio. The micro Raman microscope with high temporal and spatial resolution of the present invention realizes high-resolution acquisition and / or rapid imaging of Raman signals in deep tissues of living animals, and the Raman imaging speed is increased by 1000 times to the second level; the spectral resolution is increased by 100 times to 0.001nm. This application function is not available in current commercial Raman instruments. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without paying any creative work.

[0041] Figure 1 These are the Raman spectra of (a) rhodamine B, (b) p-mercaptobenzoic acid, and (c) cyanine 5 collected in Example 1 under 785 nm wavelength laser excitation.

[0042] Figure 2 The Raman spectrum and characteristic Raman peak of (a) p-mercaptobenzoic acid collected from neuronal cells under 785 nm laser excitation in Example 2; (b) 1082 cm -1 Cell imaging at 1588cm -1 Cell imaging at .

[0043] Figure 3 The wavelength of the laser excitation in Example 3 is 1588cm -1 (a) Raman imaging of brain slices collected at the peak position, and the corresponding Raman spectra of different brain regions (b) cortex; (c) hippocampus; (d) thalamus.

[0044] Figure 4 It is a schematic diagram of the structure of a micro Raman microscope with high temporal and spatial resolution according to the present invention.

[0045] Figure 5 It is a schematic diagram of a micro-electromechanical scanning subsystem of the present invention.

[0046] Figure 6 It is a schematic diagram of a high-speed acquisition subsystem of the present invention.

[0047] Figure 7 It is a schematic diagram of Raman spectrum obtained by Fourier transformation in the present invention. DETAILED DESCRIPTION

[0048] The present invention is further described in detail with reference to the following specific examples and drawings. The process, conditions, experimental methods, etc. for implementing the present invention, except for the contents specifically mentioned below, are all common knowledge and common common sense in the art and are not particularly limited by the present invention.

[0049] The present invention proposes a miniature high spatiotemporal resolution Raman microscope, a construction method and an application. First, a high spatiotemporal resolution in vivo fiber Raman imaging system is built. On this basis, a high-throughput Fourier transform data conversion analysis algorithm and Raman spectrum data accurate extraction and image reconstruction software based on a dual-comb pulse light source and coherent Raman imaging are established. After the dual-comb light source interacts with the probe, a series of beat frequency signals are generated for Fourier transform to obtain Raman spectrum data. A machine-based deep learning algorithm is developed to accurately extract characteristic spectral data, including Raman shift and Raman intensity, and through the process of gradient descent and back propagation, large-scale Raman imaging is predicted with higher accuracy, and high-resolution Raman imaging image reconstruction is achieved, thereby realizing efficient collection and / or imaging of in vitro and in vivo Raman signals. The miniature high spatiotemporal resolution Raman microscope consists of four parts, including a dual-comb excitation light source, a micro-electromechanical scanning subsystem, a high-speed signal acquisition subsystem, and a drive control and data processing subsystem. The present invention also proposes the application of the miniature high spatiotemporal resolution Raman microscope in Raman signal collection and / or imaging in solutions, neuronal cells and brain slices.

[0050] The present invention aims to achieve high temporal and spatial resolution Raman imaging of a freely moving living brain. It utilizes the tunable coherent resonant excitation of a dual-comb light source and the high-frequency scanning advantages of a micro-electromechanical system (MEMS), innovatively applies high numerical aperture microlenses, multimode optical fibers, high-speed photodetectors, and data acquisition cards, to develop a miniature Raman imaging system with high temporal and spatial resolution and high spectral resolution, and establishes scanning control software based on Raman signal acquisition and an image processing software system for Raman data processing and reconstruction, providing new imaging and analysis technology tools for relevant researchers to explore important information such as the concentration, distribution, and dynamic changes of different chemical substances in the brains of freely moving living animals.

[0051] The micro Raman microscope with high temporal and spatial resolution in the present invention comprises:

[0052] A light source part, wherein the light source part adopts a dual-comb pulse light source;

[0053] A micro-electromechanical scanning subsystem, wherein the micro-electromechanical scanning subsystem comprises a multi-mode optical fiber, a micro-electromechanical system, and a gradient refractive index lens;

[0054] A high-speed signal acquisition and storage subsystem, wherein the high-speed signal acquisition and storage subsystem comprises a short-pass filter LFP, a high-speed photodetector APD and a high-speed data acquisition card;

[0055] The drive control and data processing subsystem includes a drive control module and a data processing module; and is used to control the light source part, the micro-electromechanical scanning subsystem, the high-speed signal acquisition and storage subsystem, and process the acquired data.

[0056] Example 1: A micro Raman microscope with high temporal and spatial resolution is used to collect signals of different Raman molecules in a solution

[0057] In order to evaluate the Raman signal collection of Raman molecules in solution by a micro-high spatiotemporal resolution Raman microscope, different molecules, including rhodamine B, p-mercaptobenzoic acid, and cyanine dye 5, were first dissolved in ethanol to prepare a 1mM solution. Secondly, the frequency difference Δf of the dual light comb of the micro-high spatiotemporal resolution Raman microscope was adjusted to 1Hz, the scanning resolution was set to 512x512 pixels, and then the Raman signal was collected. The method of measuring Raman signal molecules in solution in the present invention has a higher spectral resolution than the existing instrument measurement method, and a single spectral measurement is shortened to the μs level.

[0058] like Figure 1 Shown are Raman spectra of different Raman molecules, with Raman characteristic peaks of different molecules.

[0059] Example 2: Miniature high temporal and spatial resolution Raman microscope for cell Raman signal collection and / or high resolution imaging

[0060] In order to evaluate whether a micro Raman microscope with high temporal and spatial resolution can be used to collect cell Raman signals, the p-thiobenzoic acid signal molecule was first incubated with neuronal cells for 30 minutes, and then the frequency difference Δf of the dual optical comb of the micro Raman microscope with high temporal and spatial resolution was adjusted to 1 Hz, the imaging resolution was set to 512x512 pixels, and the central wavelength of the optical comb was adjusted to >1000nm to reduce the background scattering signal of the optical fiber. A large numerical aperture (NA=0.8) gradient refractive index lens (GRIN) with a length of 1 mm and a diameter of 2.0 mm was used. The GRIN lens focused the excitation light onto the cells for Raman molecular signal collection and / or cell Raman imaging. Figure 2 As shown, the miniature high spatiotemporal resolution Raman microscope can be used for the collection of Raman signals of probes inside neuronal cells and / or Raman imaging of cells. This is the first reported device that can achieve high-resolution collection and rapid imaging of cell Raman signals. The Raman imaging speed is increased by 1000 times to seconds, and the spectral resolution is increased by 100 times to 0.001nm.

[0061] Example 3: Miniature high temporal and spatial resolution Raman microscope for collecting and / or imaging Raman signals in different brain regions

[0062] In order to evaluate whether a miniature high-temporal-resolution Raman microscope can be used for the collection and / or imaging of Raman signals in different brain regions, the para-mercaptobenzoic acid signal molecule was incubated with the brain slices for 30 minutes. Then the frequency difference Δf of the dual light comb of the miniature high-temporal-resolution Raman microscope was adjusted to 1 Hz, the imaging resolution was set to 1024x1024 pixels, and the central wavelength of the light comb was adjusted to >1000nm to reduce the background scattering signal of the optical fiber. A large numerical aperture (NA=0.8) gradient refractive index lens (GRIN) with a length of 5 mm and a diameter of 2.0 mm was used. The GRIN lens focused the excitation light onto the plane of the brain slice to collect and / or image the Raman signals of different brain regions in the brain slice. Figure 3 As shown, the gradient refractive index lens can be used to collect and / or image the Raman signals of probes in different brain regions. This is the first reported device that can achieve high-resolution collection and rapid imaging of Raman signals in deep brain regions.

[0063] The protection content of the present invention is not limited to the above embodiments. Without departing from the spirit and scope of the present invention, changes and advantages that can be thought of by those skilled in the art are included in the present invention and are protected by the attached claims.

Claims

1. A miniature Raman microscope with high temporal and spatial resolution, characterized in that: include: A light source part, wherein the light source part adopts a dual-comb pulse light source; A micro-electromechanical scanning subsystem, the micro-electromechanical scanning subsystem comprising: a multi-mode optical fiber, a micro-electromechanical system, and a gradient refractive index lens; A high-speed signal acquisition and storage subsystem, the high-speed signal acquisition and storage subsystem comprising: a short-pass filter LFP, a high-speed photodetector APD and a high-speed data acquisition card; The drive control and data processing subsystem includes a drive control module and a data processing module; the drive control and data processing subsystem is used to control the light source part, the micro-electromechanical scanning subsystem, the high-speed signal acquisition and storage subsystem, and process the acquired data.

2. The miniature high temporal and spatial resolution Raman microscope according to claim 1, characterized in that: The light source part includes two optical comb pulse light sources with different frequencies, which are used to generate two optical combs with fixed pulse repetition rates; the frequencies of the two optical combs are f1 and f2 respectively, and the repetition frequency difference of the beat frequency signals of the two optical combs is Δf=f2-f1.

3. The miniature high temporal and spatial resolution Raman microscope according to claim 1, characterized in that: In the micro-electromechanical scanning subsystem, the central wavelength of the multi-mode optical fiber is 1064nm, the mode field diameter is 6.7μm, the working wavelength loss is <100dB / km, and the dispersion is <1ps / nm.km; the gradient refractive index lens has a length of 1-5mm, a diameter of 0.5-2.0mm, and a numerical aperture of 0.

8.

4. The miniature high temporal and spatial resolution Raman microscope according to claim 1, characterized in that: In the high-speed signal acquisition and storage subsystem, the short-pass filter is used to filter the excitation light in the detection signal; the high-speed silicon photodetector has a variable gain and an integrated thermistor, which converts the optical signal into an electrical signal in real time; the high-speed data acquisition card is used to store and read the data.

5. The miniature high temporal and spatial resolution Raman microscope according to claim 1, characterized in that: The drive control and data processing subsystem generates a beat frequency signal after the interaction between the dual light comb light source and the probe, and performs Fourier transform to obtain Raman spectrum data; the Fourier transform data processing is performed based on the following formula: F(ω)=∫f(t)·e -iωt dt, When f(t) is a non-periodic continuous function, where F(ω) represents the frequency domain signal, f(t) represents the time domain signal, ω is the angular frequency, t is the time, and e^(-iωt) is a complex exponential function.

6. The miniature high temporal and spatial resolution Raman microscope according to claim 1, characterized in that: The miniature high-temporal-spatial resolution Raman microscope also includes a second reflector, a second dichroic mirror, a third collimator, and a multimode optical fiber; which are used to adjust the propagation path of the laser, and to collimate and transmit the laser; and / or, The light source part also includes a first collimator, a second collimator, a first reflector, and a first dichroic mirror, which are used to adjust and transmit the optical path of the excitation light.

7. A method for constructing a miniature high temporal and spatial resolution Raman microscope as claimed in any one of claims 1 to 6, characterized in that: Including construction and debugging, the construction and debugging of the hardware structure including the light source part, micro-electromechanical scanning subsystem, high-speed signal acquisition and storage subsystem, drive control and data processing subsystem, including the following steps: Step 1: Generation and tuning of the optical comb, including the following sub-steps: Step 1-1: Use a fiber femtosecond laser as a stable pump light source to generate coherent excitation; Step 1-2: Tuning is performed through the spatial optical path, so that a dual-comb light source with a fixed frequency difference Δf, adjustable intensity and spot size is finally obtained after tuning the spatial optical path; Step 2: MEMS scanning subsystem integration and debugging, including the following sub-steps: Step 2-1: Based on the sensitivity of the deformation of the piezoelectric ceramic to the applied voltage, the angle of the reflector in the micro-electromechanical system is changed by changing the applied voltage to achieve control of the scanning optical path; Step 2-2: Use a high numerical aperture gradient refractive index lens to focus the excitation light onto the scanning plane to achieve high-resolution imaging under the electrical drive signal; Step 2-3: Use multimode optical fiber as the transmission medium of pulsed laser to reduce the nonlinear broadening and dispersion of the excitation light and ensure the excitation efficiency; Step 3: Light path adjustment and calibration, including the following sub-steps: Step 3-1: Use two optical combs with a repetition frequency difference of Δf as pulse light sources. After passing through the second dichroic mirror, they are focused by the collimator and enter the multimode optical fiber, and then enter the micro-electromechanical scanning subsystem at the other end of the optical fiber. Δf = f2-f1, where f1 and f2 are the frequencies of the two optical combs respectively. Step 3-2: The excitation light and the detection light are located at the same end of the optical fiber, the micro-electromechanical scanning subsystem is located at the other end of the optical fiber, and both ends of the optical fiber are fixed on the optical platform to ensure the effective transmission of the optical signal.

8. An application of a micro Raman microscope with high temporal and spatial resolution constructed according to the method of claim 7 in collecting Raman molecular signals in vitro under excitation light; wherein: The Raman molecules include rhodamine B, p-mercaptobenzoic acid, and cyanine dye 5; The excitation light wavelength is selected from one of 633nm and 785nm; The Raman molecule concentration is 0.1-5mM; Scanning range: 100-3200cm -1 .

9. An application of a micro Raman microscope with high temporal and spatial resolution constructed according to the method of claim 7 in the collection and / or imaging of intracellular Raman molecular signals under excitation light; wherein: The cells are neuronal cells; The excitation light wavelength is selected from one of 633nm and 785nm; The Raman molecules include rhodamine B, p-mercaptobenzoic acid, and cyanine dye 5; Scanning range: 100-3200cm -1 .

10. An application of a miniature high spatiotemporal resolution Raman microscope constructed according to the method of claim 7 in the collection and / or imaging of Raman molecular signals in a mouse brain slice under excitation light; wherein: The excitation light wavelength is selected from one of 633nm and 785nm; The Raman molecules include rhodamine B, p-mercaptobenzoic acid, and cyanine dye 5; Scanning range: 100-3200cm -1 .

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