WO2021228187A1 - 脉冲型延时色散光谱测量方法和装置及光谱成像方法和装置 - Google Patents

脉冲型延时色散光谱测量方法和装置及光谱成像方法和装置 Download PDF

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WO2021228187A1
WO2021228187A1 PCT/CN2021/093540 CN2021093540W WO2021228187A1 WO 2021228187 A1 WO2021228187 A1 WO 2021228187A1 CN 2021093540 W CN2021093540 W CN 2021093540W WO 2021228187 A1 WO2021228187 A1 WO 2021228187A1
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module
spectrum
time
photons
sample
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French (fr)
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王平
李昊政
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华中科技大学
武汉华眸光电科技有限公司
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/65—Raman scattering
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/01—Arrangements or apparatus for facilitating the optical investigation
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/65—Raman scattering
    • G01N21/658—Raman scattering enhancement Raman, e.g. surface plasmons
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/01—Arrangements or apparatus for facilitating the optical investigation
    • G01N2021/0106—General arrangement of respective parts
    • G01N2021/0112—Apparatus in one mechanical, optical or electronic block
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/65—Raman scattering
    • G01N2021/653—Coherent methods [CARS]

Definitions

  • the invention belongs to the technical field of spectrum measurement and spectrum imaging, and more specifically, relates to a pulse-type time-lapse dispersion spectrum measurement method and device, and a spectrum imaging method and device.
  • Fluorescence spectroscopy originates from the transition of electronic states in atoms and molecules, and can be applied to imaging, atomic and molecular composition, state recognition, and fluorescent labeling.
  • the line width of the fluorescence spectrum is relatively wide, mostly in the range of 20 nm to 100 nm, which makes it difficult for the fluorescence imaging technology to achieve more than five kinds of multi-color imaging.
  • Raman spectroscopy is derived from the vibrational dynamics of molecules. Its Raman fingerprint has good molecular specificity, can identify and characterize chemical components in complex systems, and can quantify the concentration of molecules.
  • Raman spectrum has a narrower characteristic peak line width, only 0.1nm ⁇ 1nm, can realize multi-color Raman imaging.
  • Raman tags Bose or more colored Raman tags can be developed to realize the labeling, identification, positioning and imaging of more than one hundred proteins and ribonucleic acid (RNA) in organisms.
  • RNA ribonucleic acid
  • the detector of an ordinary Raman spectrometer usually uses a CCD camera for spectrum collection.
  • the CCD camera not only has low sensitivity and requires a complicated cooling system to suppress electronic noise, but also has a slow data transmission speed and cannot achieve high-speed signal acquisition.
  • the sensitivity of the photomultiplier tube (PMT) is much higher than that of CCD pixels, but the use of a highly sensitive photomultiplier tube array to replace CCD pixels requires 100 to 1000 independent PMT probes, which is expensive and complex to implement. Therefore, the mode of only relying on the CCD spectrometer to record the Raman spectrum greatly limits the sensitivity of Raman and restricts the development of Raman for a long time.
  • Coherent Raman scattering includes Coherent Anti-Stokes Scattering (CARS) and Stimulated Raman Scattering (SRS), and its Raman signal strength is 3-7 higher than ordinary Raman signals. Orders of magnitude. Therefore, CARS and SRS are widely used in the field of label-free bioimaging.
  • CARS uses a photomultiplier tube (PMT) and SRS uses a phase-locked amplification method to achieve Raman signal detection.
  • PMT photomultiplier tube
  • SRS uses a phase-locked amplification method to achieve Raman signal detection.
  • the advantage of both is that it can achieve high-speed and high-sensitivity Raman molecular imaging, but the disadvantage is that it can only be performed on a single Raman peak. Imaging.
  • the purpose of the present invention is to provide a pulse-type delay dispersion spectroscopy measurement method and device, which aims to solve the problem of low spectral measurement efficiency caused by insufficient detection sensitivity of the spectrometer in the prior spectroscopy measurement technology.
  • the present invention provides a pulse-type delay dispersion spectrum measurement method, which includes the following steps:
  • S2 Delay different photons in the instantaneous spectrum in time and separate them in sequence according to the time sequence;
  • S3 Use high-speed and high-sensitivity photodetectors to detect photons that come over time in the spectrum.
  • step S2 different photons in the instantaneous spectrum are delayed and dispersed in time according to wavelength, frequency, phase, energy, polarization, wave vector direction or intensity.
  • the high-speed and high-sensitivity photodetector adopts a single-channel detection element, including a photomultiplier tube, a silicon-based photomultiplier tube, a photon counter, a single photon avalanche diode, or a modem photoelectric amplification detector.
  • the present invention also provides a pulse-type delayed dispersion spectrum measuring device, which includes a pulsed laser generating module, a delayed dispersion module and a photodetection module.
  • the pulsed laser generating module is used to generate pulsed lasers and incident the pulsed lasers to the sample to be tested.
  • the upper excitation sample generates an instantaneous spectrum;
  • the time-delay dispersion module is used to delay different photons in the instantaneous spectrum and separate them in sequence according to the time sequence;
  • the photodetection module is used to detect photons in the spectrum that come with time.
  • Another object of the present invention is to provide an imaging method based on pulsed time-lapse dispersion spectroscopy, which aims to solve the technical problem of slow spectral imaging speed due to low spectral measurement efficiency in the prior art.
  • the present invention provides a spectral imaging method based on the above-mentioned spectral measurement method.
  • the invention provides a pulsed time-delay dispersion spectral imaging device, which includes: a pulsed laser generating module, a reflection module, a laser scanning module, an objective lens focusing module, a sample stage, a photon collection module, a time-delay dispersion module, a photoelectric detection module, and a collection Module;
  • the pulsed laser generator module is used to generate pulsed laser;
  • the reflection module is used to reflect the pulsed laser to the laser scanning module;
  • the laser scanning module is used to scan the laser spatially and realize imaging;
  • the objective lens focus module is used to focus the laser onto the sample;
  • the sample stage is used to place the sample and move the sample to complete the imaging;
  • the photon collection module is used to collect the instantaneous spectrum generated by the sample and output;
  • the delay dispersion module is used to delay the different photons in the instantaneous spectrum in time and follow the sequence Separate one by one;
  • the photodetection module is used to detect the photons that
  • the present invention also provides a pulse-type delay dispersion spectral imaging device, which includes: a dual-channel pulsed laser generating module, a first power adjustment module, a second power adjustment module, an optical modulation module, a time delay, a second reflection module, Dichroic mirror module, laser scanning module, objective lens focusing module, sample stage, photon collection module, time-lapse dispersion module, photodetection module and acquisition module; dual-channel pulsed laser generator module is used to generate pump light and Stokes Optical two-way pulse laser; the first power adjustment module is used to adjust the power of the Stokes light; the second power adjustment module is used to adjust the power of the pump light; the light modulation module is used to adjust the power of the Stokes light The power or phase or polarization or delay of the light is modulated; the time retarder is used to register the pulses of the pump light and the Stokes light in time; the first reflection module is used to reflect the pump light to the second Dichroic mirror module; the dichroic mirror module is used to reflect the
  • the photon collection module is a forward detection photon collection module or a backward detection photon collection module; the forward detection photon collection module is used to collect and output the instantaneous spectrum generated by the sample in the direction of penetrating the sample; and the backward detection photon collection The module is used to collect and output the instantaneous spectrum generated by the sample in the opposite direction of the excitation light.
  • the time-delay dispersion module includes a dispersion fiber, one end of which is connected to a photon collection module, and the other end is connected to a photodetection module; when photons of different wavelengths are emitted from the outlet of the dispersion fiber over time, the photodetection module treats them Perform photodetection and generate a spectrum.
  • the delay dispersion module includes: a light splitting element, a conjugate module, a beam splitting module, and a delay reflection module;
  • the light splitting element is used to split the photons collected and collimated by the photon collection module, so that The photons of different wavelengths in the spectrum are sequentially diffracted to different angles and then emitted;
  • the conjugate module is used to guide the emitted light into the reflection module;
  • the delayed reflection module is used to reflect the imported light multiple times and then return to the original path to make the spectrum Different wavelengths of photons reflect different back and forth distances to achieve different time delays for photons of different wavelengths;
  • the beam splitting module is used to transfer the returned delayed photons to the photodetection module.
  • the delay dispersion module includes: an optical wavelength splitter, an optical fiber waveguide, and an optical multiplexer; the optical wavelength splitter is used to distribute the photons collected and collimated by the photon collection module to optical fiber waveguides of different lengths; Different lengths of optical fiber waveguides make the photons of different wavelengths travel differently, and realize different time delays for photons of different wavelengths; the optical multiplexer is used to combine the time-delayed multiple photons and output them to the photodetection module.
  • the integration time of the spectral collection will be reduced as soon as it is improved, and the speed of spectral imaging will be significantly improved.
  • Raman spectroscopy the acquisition of traditional Raman spectra takes 0.1 second, and Raman spectroscopy imaging of 1000 times 1000 pixels needs to acquire 1 million Raman spectra, which requires 27.8 hours, and the imaging speed is too slow.
  • the new method can increase the speed of spectrum acquisition to 1 microsecond, so that a megapixel Raman spectrum can be completed in only 1 second.
  • the present invention has the following technical advantages:
  • the present invention concentrates the excitation laser to a very short instant pulse, uses the instant pulse to excite the sample, and concentrates the excited spectrum in the sample into the time period between the excitation light pulses, and then uses a single-channel high-sensitivity photoelectric
  • the detector achieves high-efficiency direct measurement of the time-expanded spectrum, without the need to rely on multi-channel detection equipment to measure the spectrum.
  • the invention can use a more sensitive single-channel photodetector, which not only can efficiently use all photons in the spectrum, but also can increase the sensitivity of spectrum measurement by an order of magnitude, while avoiding the need to use multi-pixels such as CCD in traditional spectrometers. , Multi-channel, low-sensitivity, low-efficiency detectors to measure the spectrum.
  • the excitation of the pulsed laser and the detection time after the pulse are one cycle, and the spectrum collected in each cycle is exactly the same.
  • the photons in the spectrum can be used for the integration of the repetitive signal, the excitation light and the generated spectral photons, both It is completely used for spectrum measurement without waste; thereby improving the photon utilization rate and improving the efficiency of spectrum measurement.
  • the structure of the spectrum measurement device provided by the present invention is simpler, and no high-precision grating is required.
  • the spectral resolution is determined by the resolution of time delay dispersion; the use of various filters is reduced, because the excitation light and the spectrum are time-dependent Separate detection, there will be no crosstalk between different wavelengths; and the use of a single-channel probe can achieve full-spectrum measurement, without segmented measurement or moving grating; excitation wavelength can be adjusted at will; the interference of background fluorescence can be eliminated through dual wavelengths , Especially suitable for application in Raman spectroscopy and CARS spectroscopy.
  • the present invention can measure low-wavenumber Raman spectroscopy. Because the traditional Raman spectrometer based on spatial dispersion needs to apply a long-pass boundary filter to suppress the scattering of the excitation laser in the spectrometer, the spectrum near the wavelength of the excitation light is cut off; therefore, the Raman spectrum with low wavenumber ( ⁇ 500cm -1 ) Usually cannot be measured.
  • the method of applying the time-delay dispersion spectroscopy of the present invention can avoid the use of filters, so that the Raman spectra with low wave numbers can be effectively collected.
  • the present invention is more advantageous to the spectrum generated by the nonlinear effect.
  • the spectrum generated by optical nonlinear effects usually requires picosecond or femtosecond pulsed laser excitation. The shorter the pulse width and the higher the power density of the pulsed light, the stronger the signal of the nonlinear spectrum.
  • the spectrum will be generated instantaneously in picoseconds or femtoseconds.
  • the time gap between pulses is used for time-delayed spectrum acquisition, the spectrum will be divided and separated, the overlap in time will be less, and the spectral resolution will be extremely high. Greatly improved.
  • the single-channel high-speed high-sensitivity photodetector in the present invention includes but is not limited to photomultiplier tube (PMT), silicon-based photomultiplier tube (SiPM), photon counter, single photon avalanche diode (SPAD), modem photoelectric Amplify the detector and so on to realize the detection of the spectrum. Because these detectors are orders of magnitude higher than traditional spectrometer cameras in terms of photoelectric amplification gain and suppressing signal noise, the present invention greatly improves the sensitivity of spectrum measurement and photon utilization.
  • the present invention uses gratings or other spatial dispersion elements in the time-delay dispersion module to diffract the photons of different wavelengths in the spectrum to different angles, and then enter the two almost parallel mirrors for multiple reflections and return to the original path.
  • the structure of the spectrometer can be greatly simplified, and the various filters required in the traditional spectrometer can be removed.
  • the use of dispersive fibers or wavelength splitters in the other two types of delay dispersion modules can further completely remove the grating, with a simpler structure, further miniaturization, and higher reliability.
  • the present invention combines various specific Raman labeled probes to target genes to a variety of specific proteins or specific RNAs of organisms, so as to realize the identification of proteome and RNA transcriptome and multi-color high-speed imaging.
  • Figure 1 is a schematic diagram of time-delayed dispersion spectroscopy measurement based on pulse excitation, in which (a) is a schematic diagram of the spectrum obtained according to the existing spatial dispersion-based spectroscopy method, and (b) is obtained according to the time-delayed dispersion spectroscopy method provided by the present invention Schematic diagram of the spectrum;
  • Figure 2 is an energy level transition diagram of the Raman process and the CARS process provided by the prior art
  • Fig. 3 is an implementation flow chart of the pulse-type delay dispersion spectroscopy measurement method provided by the present invention.
  • FIG. 4 is a block diagram of the structure and principle of the pulse-type delay dispersion spectrum measuring device provided by the present invention.
  • FIG. 5 is a schematic diagram of the structure of a pulsed excitation-based delayed dispersion spontaneous Raman spectroscopy imaging device according to an embodiment of the present invention
  • FIG. 6 is a schematic structural diagram of a time-lapse dispersion CARS spectral imaging device provided by an embodiment of the present invention.
  • Fig. 7 is a schematic diagram of CARS generated by wide-spectrum pump with opposite time dispersion and Stokes light provided by the present invention
  • Fig. 8 is a schematic structural diagram of three delay dispersion modules provided by an embodiment of the present invention, in which (a) is a schematic diagram of the principle of delay dispersion; (b) a delay based on the principle of optical fiber delay dispersion provided by the first embodiment Schematic diagram of the structure of the dispersion module; (c) The second embodiment provides a schematic diagram of the structure of the delay dispersion module based on the principle of mirror delay dispersion; (d) The third embodiment provides the realization of the principle of delay dispersion based on the wavelength divider Schematic diagram of the structure of the delay dispersion module.
  • the present invention proposes a new method and system for spectral measurement and spectral imaging based on pulse excitation and time delay dispersion, which aims to solve the problem of low spectral measurement efficiency due to insufficient detection sensitivity of the spectrometer in the existing spectral measurement and spectral imaging technology.
  • the problem of slow imaging speed The invention cleverly combines short-pulse lasers, time-delay dispersion spectroscopy, high-speed high-sensitivity detectors, breaks through the concept of traditional spatial dispersion spectrometers, realizes time-delay dispersion spectrometers in the time dimension, and can be completed with only one channel of high-sensitivity probes. Spectral measurement.
  • the method of time-delayed dispersion spectroscopy measurement gets rid of the predicament that existing spatial dispersion spectrometers can only use low-efficiency linear array cameras or low-sensitivity photoelectric conversion arrays to obtain spectra, and can use high-gain or single-photon detectors to greatly increase the measurement sensitivity.
  • This method first uses short pulse light to excite the sample to generate an instantaneous spectrum, then separates the photons in the spectrum in time according to wavelength or other spectral characteristics, and finally uses the photon detection advantage of a single high-speed and high-sensitivity photodetector to detect random changes in the spectrum. Different photons from time.
  • the present invention provides a pulse-type delay dispersion spectrum measurement method, which includes the following steps:
  • S2 Delay different photons in the instantaneous spectrum in time and separate them in sequence
  • S3 Use high-speed and high-sensitivity photodetectors to detect photons that come over time in the spectrum.
  • a short pulse laser (specifically, a pulsed laser with a pulse width time of 1 microsecond to 1 attosecond) generates an instantaneous spectrum (1 microsecond to 1 attosecond) when it interacts with the sample, and the spectrum includes fluorescence.
  • Spectroscopy used to analyze the electronic state energy level transitions of molecular atoms and molecules
  • Raman scattering spectroscopy used to analyze the vibrational energy level transitions of molecules
  • absorption spectroscopy used to analyze the vibrational energy level transitions of molecules
  • Other spectra including but not limited to rotational spectroscopy, infrared absorption spectroscopy, etc., which can help analyze the chemical and physical properties of atoms and molecules).
  • step S2 different photons in the instantaneous spectrum are delayed and dispersed back and forth in time in the order of wavelength or other spectral characteristics (energy, frequency, intensity, polarization, wavefront, phase, wave vector direction), until the next The laser pulse arrives.
  • the spectrum uses the time gap between the excitation pulses to expand enough time.
  • photons of different wavelengths or other spectral characteristics reach the detector, while ensuring that the high-speed photodetector can sequentially detect different wavelengths or spectra according to time.
  • the characteristic photons are then deduced from the spectrum based on the arrival time.
  • a single-channel high-speed and high-sensitivity photodetector can be used to detect photons of different wavelengths or spectral characteristics arriving at different delays.
  • Single-channel high-speed high-sensitivity photodetectors include, but are not limited to, photomultiplier tubes (PMT), silicon-based photomultiplier tubes (SiPM), photon counters, single photon avalanche diodes (SPAD), modem photo-amplified detectors, etc.
  • the single pulse excitation and the detection time after the pulse are one cycle, and the spectrum collected in each cycle is exactly the same, which can be used for the integration of repetitive signals.
  • the repetition frequency of a pulsed laser is the number of spectral detections per second.
  • the pulse and signal acquisition need to be accurately synchronized, and the Raman spectrum after the pulse can be accurately superimposed and integrated.
  • the pulse laser outputs the trigger signal of the laser pulse to the time-delay spectrum acquisition card in real time to synchronize the laser pulse and spectrum acquisition.
  • the invention avoids the disadvantage that the pixel array in the low-sensitivity camera must be used as the spectrum detector in the traditional spectrometer, and opens up a time-delay dispersion spectroscopy method in the time domain, and uses a single-channel high-sensitivity detector to detect the spectrum in time sharing. Because the instantaneous pulsed laser excitation spectrum is used in the present invention, after delay dispersion, different wavelengths are obtained by time sharing. Therefore, in the present invention, the method of time-sharing detection of different wavelengths in the spectrum does not have the problem of low duty cycle and wasting photons as in the usual time-sharing multiplexing method. Instead, it avoids the single-channel probe that only detects one wavelength and wastes the remaining wavelengths. The problem.
  • the present invention also provides a pulse-type delay dispersion spectrum measurement device, including: a pulsed laser generator module, a delay dispersion module and a photodetection module.
  • the laser generator module is used to generate short pulses with a wavelength of ⁇ l.
  • the time-delay dispersion module is used to delay different photons in the instantaneous spectrum in time and separate them in sequence; photodetection module It is used to detect the photons that come over time in the spectrum.
  • the time-delay dispersion module delays and combines different photons in the instantaneous spectrum in the order of wavelength or other spectral characteristics (energy, frequency, intensity, polarization, wavefront, phase, and wave vector direction) in time. Disperse until the next laser pulse arrives.
  • the delay generated by photons of different wavelengths or spectral characteristics in the delay dispersion unit corresponds to its wavelength in a one-to-one correspondence.
  • a dispersion fiber can be used in the delay dispersion module to delay photons of different wavelengths in the spectrum, and the exit port of the fiber ensures that the high-speed detector detects photons of different wavelengths sequentially in time.
  • a grating can be used in the time-delay dispersion module to diffract photons of different wavelengths in the spectrum to different angles, and then enter two almost parallel mirrors for multiple reflections and then return to the original path. Raster. Because photons of different wavelengths in the spectrum travel differently back and forth between the mirrors, photons of different wavelengths produce different time delays. Ensure that the high-speed detector detects photons of different wavelengths in sequence at the light exit port according to time.
  • a wavelength splitter can be used in the delay dispersion module to distribute photons of different wavelengths in the spectrum to different optical fiber waveguides. Due to the different lengths of the optical fiber waveguides, the propagation distances of photons of different wavelengths are different. Finally, the different paths of light are combined and output through the optical multiplexer, so that photons of different wavelengths in the spectrum have different delays, ensuring that the high-speed detectors sequentially detect photons of different wavelengths according to time.
  • the short pulse laser generated by the laser generation module can excite the instantaneous fluorescence spectrum in the sample; the time-delay dispersion module delays and disperses different photons in the instantaneous fluorescence spectrum in the order of wavelength until The next laser pulse arrives.
  • Fluorescence spectroscopy uses the time gap between excitation pulses for sufficient time expansion to ensure that high-speed photodetectors can sequentially detect photons of different wavelengths in time.
  • the short pulse laser generated by the laser generating module can excite the instantaneous spontaneous Raman scattering spectrum in the sample.
  • the delayed dispersion module delays and disperses different photons in the instantaneous spontaneous Raman scattering spectrum in the order of wavelength in time before and after, until the next laser pulse arrives.
  • Raman spectroscopy takes advantage of the time gap between excitation pulses for sufficient time expansion to ensure that high-speed photodetectors can sequentially detect photons of different wavelengths in time.
  • the advantage of the present invention on the measuring device is that it not only realizes the improvement of the spectral detection sensitivity in the order of magnitude, but also simplifies the structure of the spectrometer, the whole machine can be further miniaturized, the transportability is improved, and the manufacturing cost is reduced.
  • the present invention also provides a spectral imaging method based on pulse-type time-delay dispersion spectroscopy.
  • the imaging method can solve the technical problem of slow spectral imaging speed due to low spectral measurement efficiency in the prior art because of the adoption of the previous technique.
  • the spectral measurement method described in the article is due to the fact that the spectral measurement method can improve the efficiency of spectral measurement, so that the imaging method of the present invention has the advantage of fast imaging speed.
  • the imaging method can be realized by conventional technical means such as moving the sample stage or scanning the sample with a laser for imaging. As for the specific implementation steps of the imaging method, it is the prior art, and will not be repeated here.
  • the spectral imaging mode of the sample in the three-dimensional space can be applied but not limited to three-dimensional laser scanning or three-dimensional sample translation stage.
  • the present invention can also be combined with Surface Enhanced Raman Scattering (SERS) to realize more sensitive Raman spectrum detection and imaging, and realize the separation of Raman spectrum and background spectrum.
  • SERS Surface Enhanced Raman Scattering
  • the spectral imaging method provided by the embodiments of the present invention can also be combined with specific Raman tags, including but not limited to molecules similar to HBI in the GFP protein structure generated by genetic modification; having carbon-carbon double bonds or carbon-carbon triple bonds or other Dye molecules with strong ⁇ -bonded Raman signals, such as rhodamine 800 or similar molecules, etc.; genes are targeted to a variety of specific proteins or specific RNAs of organisms to realize proteome and RNA transcriptome recognition and multi-color imaging .
  • specific Raman tags including but not limited to molecules similar to HBI in the GFP protein structure generated by genetic modification; having carbon-carbon double bonds or carbon-carbon triple bonds or other Dye molecules with strong ⁇ -bonded Raman signals, such as rhodamine 800 or similar molecules, etc.; genes are targeted to a variety of specific proteins or specific RNAs of organisms to realize proteome and RNA transcriptome recognition and multi-color imaging .
  • the pump light (Pump) and the Stokes light (Stokes) are short pulse lasers, which overlap in space and time, and the transient anti-Stokes Raman in the sample is excited due to the nonlinear effect.
  • Photon Coherent Anti-Strokes Raman Scattering, CARS
  • the delayed dispersion module delays and disperses different photons in the instantaneous CARS spectrum in the order of wavelength in time until the next laser pulse arrives.
  • CARS spectroscopy uses the time gap between excitation pulses for sufficient time expansion to ensure that high-speed photodetectors can detect photons of different wavelengths sequentially in time.
  • Pump light wide-linewidth femtosecond laser, or supercontinuum femtosecond laser
  • Stokes light is a narrow-band pulsed laser, which can realize CARS spectroscopy.
  • Example 1 Time-lapse dispersion spontaneous Raman spectroscopy measurement and spectral imaging based on pulse excitation.
  • Figure 1 is a schematic diagram of time-delayed dispersion spectroscopy measurement based on pulse excitation, in which (a) is a schematic diagram of the spectrum obtained according to the existing spatial dispersion-based spectroscopy method, and (b) is the time-lapse dispersion spectroscopy method provided according to the present invention Schematic diagram of the spectrum; Figure (a) can be seen: the spectrum is spatially dispersed and separated, and the many pixels of the camera array using the spectrometer are detecting different components of the spectrum, but the sensitivity of the array pixels is generally very low, and the photon utilization rate Poor.
  • the spectrum is separated in the time dimension, the dotted line is the short pulse of excitation light, and the solid line is the time-delayed spectrum.
  • the high-speed and high-sensitivity single-channel photoelectric probe can detect the delay in time sequence.
  • the time-delay dispersion spectrum can be measured and integrated repeatedly in time.
  • the repetitive sequence of the short-pulse laser excites the sample to generate the instantaneous spectrum
  • the delay dispersion module is used to delay the photons of different wavelengths in the time gap between the excitation pulses in the order of wavelength and to separate them in sequence, and finally use
  • the high-speed and high-sensitivity photodetector detects photons of different wavelengths sequentially according to time, and then deduces the spectrum according to the arrival time.
  • the single pulse excitation and the detection time after the pulse are one cycle, and the spectrum collected in each cycle is exactly the same, which can be used for the integration of repetitive signals.
  • the repetition frequency of a pulsed laser is the number of spectrum detections per second.
  • the pulse and signal acquisition need to be accurately synchronized, so that the spectrum after the pulse can be accurately superimposed and integrated.
  • FIG. 2 shows the energy level transition diagrams of the Raman process and the CARS process provided by the prior art.
  • the Raman process is: the molecules in the sample are excited by laser to generate Raman scattered photons;
  • the CARS process is: the molecules in the sample
  • the phase-locked pulsed laser pumps Pump light and Stokes light (the energy difference between the two photons is equal to the transition energy of the molecular ground state) to generate anti-Stokes photons.
  • the energy of the scattered photon and the photon energy of the excitation light differ by one molecular vibrational energy level. Therefore, the species and relative concentration of molecules in the sample can be identified by detecting the spectra of Raman scattered photons.
  • the embodiment of the present invention also provides a time-delayed dispersive Raman spectroscopy measurement and spectral imaging device based on pulse excitation.
  • various system parameters in the device can be selected according to actual conditions.
  • the pulsed time-delay dispersive spectral imaging device includes: a pulsed laser generating module 1, a first reflection module 102, a laser scanning module 103, an objective lens focusing module 104, a sample stage 105, a photon collection module 106, and a time-lapse dispersion Module 2, photodetection module 3, and acquisition module 4; pulse laser generation module 1 is used to generate pulse laser; first reflection module 102 is used to reflect pulse laser to laser scanning module 103; laser scanning module 103 is used to scan in space Laser and realize imaging; the objective lens focusing module 104 is used to focus the laser onto the sample; the sample stage 105 is used to place the sample; the photon collection module 106 is used to collect the instantaneous spectrum generated by the sample and collimate it for output; the delay dispersion module 2 It is used to delay processing the different photons in the instantaneous spectrum in time and sequentially separate them according to the time sequence; the photodetection module 3 is used to detect the photo
  • the working process of the imaging device is as follows: the pulsed laser enters the three-dimensional or two-dimensional laser scanning module 103 and the objective lens focusing module 104 of the imaging microscope through the first reflection module 102 to complete the laser scanning and imaging of the sample.
  • the laser scanning system can be a combination of a scanning galvanometer and other laser scanning methods.
  • the three-dimensional displacement sample stage 105 can also complete three-dimensional scanning of the sample.
  • the Raman photons generated by the interaction between the laser and the sample pass through the photon collection module 106 and then enter the time-delay dispersion module 2 (see Example 3 and FIG. 8 for details).
  • the single-channel high-speed and high-sensitivity detector performs photoelectric detection, and the acquisition module 4 transmits the spectrum to a computer or other data terminal.
  • the photon collection module 106 can be a forward detection photon collection module, or a backward detection photon collection module (the dotted line in FIG. 5); the forward detection photon collection module is used to pass through the sample The instantaneous spectrum generated by the sample is collected and output in the direction of the excitation light; the backward detection photon collection module is used to collect and output the instantaneous spectrum generated by the sample in the opposite direction of the excitation light.
  • the pulsed laser spontaneous Raman light source can emit laser pulses with a pulse width of 1 ns with a repetition frequency of 1 MHz, the average power of the laser is 100 mW to 200 mW, and the wavelength is 532 nm.
  • the pulsed laser passes through the first reflection module 102 and enters the three-dimensional or two-dimensional laser scanning module 103 and the objective lens focusing module 104 of the imaging microscope to complete the laser scanning and imaging of the sample.
  • the laser scanning system can be a combination of a scanning galvanometer and other laser scanning methods.
  • the three-dimensional displacement sample stage 105 can also complete three-dimensional scanning of the sample.
  • the Raman photons generated by the interaction between the laser and the sample pass through the photon collection module 106, which can be a confocal collection system or an optical fiber collection system, and enter the core component delay dispersion module 2 (see Example 3 and Figure 8 for details), complete Delay dispersion of different wavelength components in Raman photons. You can also enter the delay dispersion module through backward detection (the dotted line in Figure 5). Because the repetition frequency of the laser pulse is 1 MHz, the time gap between each pulse is 1000 ns.
  • the time delay dispersion module divides the collected Raman Stokes photons, the wavelength is usually 533nm ⁇ 680nm, which covers the Raman shift of 0 ⁇ 4000cm -1 , and the delay is divided into 1000 segments in 1000ns.
  • photons of different wavelengths enter the single-channel high-speed and high-sensitivity detector according to wavelength and time for photoelectric detection.
  • the adoption rate of the detector is 1 GHz, and one wavelength is sampled every 1 ns. Therefore, a Raman spectrum can be collected every 1 microsecond from the excitation of each pulse. Through precise timing control, 1 million Raman spectra can be collected in one second for integration and superposition, and a Raman spectrum with a higher signal-to-noise ratio can be obtained.
  • the spectrum is transmitted to a computer or other data terminal through the acquisition module 4. Similarly, you can wait for the completion of the spectrum collection after each pulse or multiple pulses, move the laser to a new position, and collect a new Raman spectrum, then the Raman spectrum sample imaging can be realized.
  • the Raman spectrum sample imaging can be realized.
  • only a single-channel highly sensitive probe realizes spectrum measurement and collection, and the utilization efficiency of Raman photons and the imaging speed of Raman spectrum can be greatly improved.
  • This example can also be applied to fluorescence spectrum measurement and fluorescence spectrum imaging.
  • Example 2 Time-lapse dispersion CARS spectral measurement and CARS spectral imaging example.
  • the embodiment of the present invention provides a method for time-delay dispersion CARS spectrum measurement and CARS spectrum imaging. Please refer to FIG. 6, and various system parameters can be selected according to actual conditions.
  • the pulsed time-delay dispersive spectral imaging device includes: a dual-channel pulsed laser generator module 200, a first power adjustment module 201, a second power adjustment module 202, an optical modulation module 203, a time delay 204, and a second Reflection module 206, dichroic mirror module 205, laser scanning module 103, objective lens focusing module 104, sample stage 105, photon collection module 106, time-delay dispersion module 2, photodetection module 3 and acquisition module 4; dual-channel pulsed laser generation
  • the module 200 is used to generate two pulse lasers of pump light and Stokes light;
  • the first power adjustment module 201 is used to adjust the power of the Stokes light;
  • the second power adjustment module 202 is used to adjust the power of the pump light Power level;
  • the optical modulation module 203 is used to modulate the power or phase or polarization or delay of the Stokes light;
  • the time delay 204 is used to match the pulses of the pump light and the Stokes light in time
  • the working process of the imaging device is as follows: a dual-channel pulsed CARS laser source emits two femtosecond lasers: pump light ⁇ Pump and Stokes light ⁇ Stokes .
  • the Stokes pulse laser is passed through the first power adjustment module 201
  • the light modulation module 203 modulates.
  • the time delay 204 can adjust the time delay of the Stokes light.
  • the dichroic mirror module 205 completely combines the Stokes light and the pump light in space.
  • the time delay 204 and the dichroic mirror module 205 are two This is used to ensure that the Stokes light and the pump light pulse completely coincide in time and space.
  • Two femtosecond lights enter the three-dimensional or two-dimensional laser scanning module 103 and the objective lens focusing module 104 of the imaging microscope to complete the laser scanning and imaging of the sample.
  • the three-dimensional displacement sample stage 105 can also complete three-dimensional scanning of the sample.
  • the CARS photons generated by the interaction between the laser and the sample enter the time-delay dispersion module 2 after passing through the photon collection module 106.
  • the core component delay dispersion module 2 (see Example 3 and Figure 8 for details), completes the delay dispersion of different wavelength components in Raman photons. After the delay, photons of different wavelengths enter the single-channel high-speed high-sensitivity detector 3 according to wavelength and time for photoelectric detection. Finally, the spectrum is transmitted to a computer or other data terminal through the acquisition module 4.
  • the photon collection module 106 can be a forward detection photon collection module, or a backward detection photon collection module (the dotted line in FIG. 6), and the forward detection photon collection module is used to transmit the sample The instantaneous spectrum generated by the sample is collected and output in the direction of the excitation light; the backward detection photon collection module is used to collect and output the instantaneous spectrum generated by the sample in the opposite direction of the excitation light.
  • the dual-channel pulsed CARS laser light source can emit two femtosecond lasers with a repetition frequency of 1MHz and phase-locked, pump light ⁇ Pump and Stokes light ⁇ Stokes .
  • the average power of the laser is 100mW ⁇ 1000mW, and the wavelengths are respectively Supercontinuum 800nm ⁇ 960nm and narrowband 1040nm.
  • the Stokes pulse laser is modulated by the light modulation module 203 after passing through the first power adjustment module 201.
  • the time delay 204 can adjust the time delay of the Stokes light.
  • the dichroic mirror module 205 completely combines the Stokes light and the pump light in space.
  • the time delay 204 and the dichroic mirror module 205 are two This is used to ensure that the Stokes light and the pump light pulse completely coincide in time and space.
  • Two femtosecond lasers enter the three-dimensional or two-dimensional laser scanning module 103 and the objective lens focusing module 104 of the imaging microscope to complete the laser scanning and imaging of the sample.
  • the laser scanning system can be a combination of a scanning galvanometer and other laser scanning methods.
  • the three-dimensional displacement sample stage 105 can also complete three-dimensional scanning of the sample.
  • the CARS photons generated by the interaction between the laser and the sample enter the delay dispersion module 2 through the photon collection module 106 (see Example 3 and FIG. 8 for details) to complete the delay dispersion of the different wavelength components in the Raman photons.
  • the delay dispersion module can also enter the delay dispersion module through backward detection (the dotted line). Because the repetition frequency of the laser pulse is 1 MHz, the time gap between each pulse is 1000 ns.
  • the time delay dispersion module divides the collected Raman Stokes photons, the wavelength is usually 650nm ⁇ 891nm, which covers the Raman displacement of 2885cm -1 ⁇ 801cm -1 , and the delay is divided into 1000 segments in 1000ns.
  • photons of different wavelengths enter the single-channel high-speed and high-sensitivity detector according to wavelength and time for photoelectric detection.
  • the adoption rate of the detector is 1 GHz, and one wavelength is sampled every 1 ns.
  • a Raman spectrum can be collected every 1 microsecond from the excitation of each pulse. Through precise timing control, 1 million Raman spectra can be collected in one second for integration and superposition, and a Raman spectrum with a higher signal-to-noise ratio can be obtained. Finally, the spectrum is transmitted to a computer or other data terminal through the acquisition module 4. Similarly, you can wait for the completion of the spectrum collection after each pulse or multiple pulses, move the laser to a new position, and collect a new Raman spectrum, then the Raman spectrum sample imaging can be realized. In the present invention, only a single-channel highly sensitive probe realizes CARS spectrum measurement and collection, and both the utilization efficiency of CARS photons and the imaging speed of Raman spectrum can be greatly improved.
  • pump light Puls
  • Stokes light can also be wide linewidth femtosecond lasers (10fs), or both are supercontinuum femtosecond lasers, but the center wavelengths of the two are different. Covering the vibrational spectrum of the molecule, and the time dispersion is just the opposite, as shown in Figure 7, the CARS spectrum can also be generated. Both the pump light and the Stokes light are wide linewidth pulse lasers. The photon energy difference between the two covers the molecular vibration spectrum ⁇ i , and the time dispersion is exactly the opposite, ensuring that only one molecular vibration is excited to produce CARS at a time Photon.
  • Example 3 Examples of the specific structures of the three delay dispersion modules.
  • the embodiment of the present invention provides three examples of generating delayed dispersion spectra. Please refer to Figure 8, and various system parameters and methods can be selected according to actual conditions. The principle is shown in Figure 8(a). The instantaneous spectral wavelengths generated by the pulsed laser are superimposed in time. Using the method of delayed dispersion, the photons in the spectrum can be separated in time and wavelength and detected.
  • FIG 8(b) shows the structure of the delay dispersion module based on the principle of fiber delay dispersion provided by the first embodiment.
  • the fiber delay dispersion module includes: a dispersion fiber 21, one end of which is connected to the photon collection module 106, and the other end Connect the photodetection module 3; the photon collection module 106 collects and couples all photons into the dispersion fiber 21.
  • the high-speed and high-sensitivity photoelectric detection module 3 performs photoelectric detection on them to generate a spectrum.
  • the method is simple and straightforward and does not require gratings or other dispersive elements.
  • Fig. 8(c) shows the structure of the delay dispersion module based on the principle of mirror delay dispersion provided by the second embodiment.
  • the mirror delay dispersion module includes: a light splitting element 22 and a conjugate module 23 arranged in sequence The beam splitting module 24 and the delay reflection module 25; the beam splitting element 22 is used to split the photons collected by the photon collection module 106, so that photons of different wavelengths in the spectrum are diffracted to different angles and then emitted; the conjugate module 23 is used To guide the outgoing light into the time-delayed reflection module 25; the time-delayed reflection module 25 is used to reflect the imported light multiple times and then return to the original path, so that the difference in the path difference between the reflected back and forth of photons of different wavelengths in the spectrum is realized.
  • the photons of wavelengths produce different time delays; the beam splitting module 24 is used to transfer the returned delayed photons to the photodetection module 3.
  • the distance between the beam splitting element 22 and the conjugate module 23 is the focal length of the conjugate module 23.
  • the conjugate module 23 includes two lenses, and the distance between the two lenses is the sum of the focal lengths of the two lenses.
  • the beam splitting module 24 is arranged at any position between the light splitting element 22 and the delay reflection module 25.
  • the light splitting element 22 may be a spatial dispersion element such as a grating, a prism, etc. Taking the grating as an example, the working process of the delay dispersion module of the mirror is described as follows:
  • the photon collection module 106 collects and collimates the photons, all the photons enter the grating or other spatial dispersive elements to be split, and the photons of different wavelengths in the spectrum are sequentially diffracted to different angles and emitted. Then, through the conjugate module 23 composed of two lenses, the light is guided into the two almost parallel time-lapse reflection modules 25 for multiple reflections and then returns to the original path.
  • the angle between the two reflecting plates in the time delay reflection module 25 is 0.001 degree to 1 degree; preferably, the angle is 0.01 degree.
  • photons of different wavelengths in the spectrum travel differently back and forth between the mirrors, photons of different wavelengths produce different time delays.
  • the delayed photons return, they are transferred to the photodetection module 3 by the beam splitting module 24 for detection.
  • the high-speed detector detects photons of different wavelengths in sequence at the light exit port according to time. In this specific example, a greater delay can be achieved through the optical path difference of different photons.
  • FIG. 8(d) shows the structure of the delay dispersion module based on the principle of delay dispersion of the wavelength divider provided by the third embodiment.
  • the delay dispersion module of the wavelength divider includes: an optical wavelength splitter 26 and an optical fiber waveguide arranged in sequence 27 and the optical multiplexer 28; the photon collection module 106 collects and couples all photons into the optical wavelength splitter 26.
  • the optical wavelength splitter 26 distributes the photons of different wavelengths in the spectrum to the optical fiber waveguides 27 of different lengths; due to the different lengths of the optical fiber waveguides, the propagation distances of the photons of different wavelengths are different, and finally the different paths of light pass through the optical multiplexer 28.
  • the combined output is given to the photoelectric detection module 3.
  • the optical wavelength splitter 26 can divide photons of different wavelengths into 100-1000 channels.
  • 100 to 1000 optical fiber waveguides with different lengths can be provided in the optical fiber waveguide 27.
  • the optical fiber delay dispersion module in the present invention has a simple structure, requires fewer components, and can miniaturize the instrument as much as possible; the reflector delay dispersion module and the wavelength divider delay dispersion module can provide more dispersion delay, Conducive to the improvement of spectral resolution.
  • the single-channel high-speed high-sensitivity photodetection module 3 in all specific examples includes, but is not limited to, a photomultiplier tube (PMT), a silicon-based photomultiplier tube (SiPM), a photon counter, and a single photon avalanche diode (SPAD). ), modulation and demodulation photoelectric amplifier detector, etc.
  • PMT photomultiplier tube
  • SiPM silicon-based photomultiplier tube
  • PAD single photon avalanche diode
  • Single-channel detectors have higher photoelectric sensitivity and data transmission speed than multi-channel detectors, and can also have a larger detection area to improve photon detection efficiency.

Abstract

一种脉冲型延时色散光谱测量方法和装置及光谱成像方法和装置；光谱测量方法包括：S1：通过脉冲激光激发待测样品并产生瞬时光谱；S2：将瞬时光谱中的不同光子在时间上进行延时处理并根据时序先后依次分开；S3：采用高速高灵敏光电探测器探测光谱中随时间而来的光子。采用瞬间脉冲来激发样品，并将样品中被激发出的光谱集中到激发光脉冲之间的时间段，再利用单通道高灵敏光电探测器对时间上展开的光谱实现高效率的直接测量，不需要依赖多通道探测设备测量光谱；不仅可以高效利用光谱中的所有光子，而且还可以从数量级上提高光谱测量的灵敏度，从而提高光谱测量效率。

Description

脉冲型延时色散光谱测量方法和装置及光谱成像方法和装置 【技术领域】
本发明属于光谱测量和光谱成像技术领域，更具体地，涉及一种脉冲型延时色散光谱测量方法和装置及光谱成像方法和装置。
【背景技术】
光谱因为具有分子特异性以及其它分析和测量优势，已经在生物、化学、材料、医学等众多领域以及工业生产中得到了广泛应用。荧光光谱源于原子分子中电子态跃迁，可应用于成像、原子分子成分、状态的识别、以及荧光标记。但荧光光谱谱线的线宽较宽，大都在20nm～100nm的范围，导致荧光成像技术很难实现五种以上的多色成像。相比之下，拉曼光谱源于分子的振动态跃迁，其拉曼指纹谱具有很好的分子特异性，能够识别和表征复杂系统中的化学组分，可以定量分子的浓度。并且拉曼光谱具有更窄的特征峰线宽，仅有0.1nm～1nm，可以实现多色拉曼成像。利用特别的拉曼标签，可发展百色或更多色的拉曼标记，实现对生物体中百种以上蛋白、核糖核酸(Ribonucleic acid，RNA)的标记、识别、定位与成像。但是由于传统拉曼的信号十分微弱，且生物体内的分子浓度较低，因此普通拉曼很难在生物成像上有更广阔的应用。
普通拉曼光谱仪的探测器通常采用CCD相机进行光谱采集。CCD相机不但灵敏度低，需要复杂的降温系统才能压制电子噪声，而且数据传输速度慢，不能实现高速的信号采集。光电倍增管(Photomultiplier tube，PMT)灵敏度远远高于CCD像素，但是使用高灵敏光电倍增管阵列代替CCD像素，需要100～1000个独立的PMT探头，价格昂贵，系统复杂，无法实现。因此仅依靠CCD光谱仪进行拉曼光谱记录的模式极大限制了拉曼的灵敏度，长期制约了拉曼的发展。
相干拉曼散射包括相干反斯托克斯散射(Coherent Anti-Stokes Scattering,CARS)和受激拉曼散射(Stimulated Raman Scattering,SRS)，其拉曼信号强度比普通的拉曼信号高3～7个数量级。因此CARS和SRS被广泛应用于无标记的生物成像领域。CARS通过光电倍增管(PMT)，SRS通过锁相放大方式实现拉曼信号的探测，两者的优势是都可实现高速高灵敏的拉曼分子成像，但劣势是只能针对单个拉曼峰进行成像。但对于探测更多特征峰的拉曼光谱的采集与成像，SRS则需要通过扫描脉冲激光波长等方式实现，速度慢，且光谱范围有限。同样，CARS光谱的采集也离不开传统CCD光谱仪，灵敏度不仅低而且采集速度慢。由于在光谱探测中一直无法使用量子效率或者增益放大高的探测器，导致拉曼光子的探测非常低效，意味着光子数的严重浪费。即使使用单通道PMT或光子计数器，分时探测用光栅在空间上分开的多个波长的光子，同样也会导致光子的浪费。
【发明内容】
针对现有技术的缺陷，本发明的目的在于提供一种脉冲型延时色散光谱测量方法及装置，旨在解决现有光谱测量技术中由于光谱仪的探测灵敏度不足导致光谱测量效率低的问题。
本发明提供了一种脉冲型延时色散光谱测量方法，包括下述步骤：
S1：通过脉冲激光激发待测样品并产生瞬时光谱；
S2：将瞬时光谱中的不同光子在时间上进行延时处理并根据时序先后依次分开；
S3：采用高速高灵敏光电探测器探测光谱中随时间而来的光子。
其中，在步骤S2中，将瞬时光谱中不同光子按波长、频率、相位、能量、偏振、波矢方向或强度在时间上进行延迟并分散开。
其中，在步骤S3中，高速高灵敏光电探测器采用单通道探测元件，包括光电倍增管、硅基光电倍增管、光子计数器、单光子雪崩二极管或调制解调光电放大探测器。
本发明还提供了一种脉冲型延时色散光谱测量装置，包括脉冲激光发生模块、延时色散模块和光电探测模块，脉冲激光发生模块用于产生脉冲激光，并将脉冲激光入射至待测样品上激发样品产生瞬时光谱；延时色散模块用于将瞬时光谱中的不同光子在时间上进行延时处理并根据时序先后依次分开；光电探测模块用于探测光谱中随时间而来的光子。
本发明的另一目的在于提供基于脉冲型延时色散光谱测量的成像方法，旨在解决现有技术中由于光谱测量效率低导致光谱成像速度慢的技术问题。
本发明提供了一种基于上述的光谱测量方法实现的光谱成像方法。
本发明提供了一种脉冲型延时色散光谱成像装置，包括：脉冲激光发生模块、反射模块、激光扫描模块、物镜聚焦模块、样品台、光子收集模块、延时色散模块、光电探测模块和采集模块；脉冲激光发生模块用于产生脉冲激光；反射模块用于将脉冲激光反射至激光扫描模块；激光扫描模块用于在空间上扫描激光并实现成像；物镜聚焦模块用于聚焦激光至样品上；样品台用于放置样品并移动样品完成成像；光子收集模块用于收集样品产生的瞬时光谱并输出；延时色散模块用于将瞬时光谱中的不同光子在时间上进行延时处理并根据时序先后依次分开；光电探测模块用于探测光谱中随时间而来的光子；采集模块用对探测的光子进行采集。
本发明还提供了一种脉冲型延时色散光谱成像装置，包括：双通道脉冲激光发生模块、第一功率调节模块、第二功率调节模块、光调制模块、时间延迟器、第二反射模块、二向色镜模块、激光扫描模块、物镜聚焦模块、样品台、光子收集模块、延时色散模块、光电探测模块和采集模块；双通道脉冲激光发生模块用于产生泵浦光和斯托克斯光两路脉冲激光；第一功率调节模块用于调节斯托克斯光的功率大小；第二功率调节模块用于调节泵浦光的功率大小；所述光调制模块用于对斯托克斯光的功率或者相位或者偏振或者延时进行调制；时间延迟器用于在时间上对泵浦光和斯托 克斯光的脉冲进行配准；第一反射模块用于对泵浦光进行反射至二向色镜模块；二向色镜模块用于对斯托克斯光进行反射，并在空间上使得泵浦光和斯托克斯光重合；激光扫描模块用于在空间上扫描激光并实现成像；物镜聚焦模块用于聚焦激光至样品上；样品台用于放置样品并移动样品完成成像；光子收集模块用于收集样品产生的瞬时光谱并输出；延时色散模块用于将瞬时光谱中的不同光子在时间上进行延时处理并根据时序先后依次分开；光电探测模块用于探测光谱中随时间而来的光子；采集模块用对探测的光子进行采集。
更进一步地，光子收集模块为前向探测光子收集模块或后向探测光子收集模块；前向探测光子收集模块用于在透过样品的方向收集样品产生的瞬时光谱并输出；后向探测光子收集模块用于在激发光反方向收集样品产生的瞬时光谱并输出。
作为本发明的一个实施例，延时色散模块包括色散光纤，其一端连接光子收集模块，另一端连接光电探测模块；当不同波长的光子在色散光纤出口随时间依次出射时，光电探测模块对其进行光电探测并产生光谱。
作为本发明的另一个实施例，延时色散模块包括：分光元件、共轭模块、分束模块和延时反射模块；分光元件用于对光子收集模块收集并准直后的光子进行分光，使得光谱中不同波长的光子依次衍射到不同的角度后出射；共轭模块用于将出射光导入反射模块中；延时反射模块用于对导入的光进行多次反射后再原路返回，使得光谱中不同波长的光子在反射来回的路程差不同实现了不同波长的光子产生不同的时间延迟；分束模块用于将返回的延时后的光子转移至光电探测模块。
作为本发明的另一个实施例，延时色散模块包括：光波分器、光纤波导和光复用器；光波分器用于将光子收集模块收集并准直后的光子分发至不同长度的光纤波导中；不同长度的光纤波导使得不同波长的光子的传播距离不同，实现了不同波长的光子产生不同的时间延迟；光复用器用于将 时间延迟后的多路光子进行合并并输出给光电探测模块。
在本发明提供的光谱成像方法中，伴随着光谱测量的探测效率和灵敏度的大幅改善，光谱的采集的积分时间会一进步降低，光谱成像的速度就会得到显著的提高。例如拉曼光谱，传统拉曼光谱的采集需要0.1秒，1000乘以1000像素的拉曼光谱成像需要采集1百万条拉曼光谱，则需要27.8小时，成像速度太慢。新的方法可将采谱速度提高到1微妙，这样一幅百万像素的拉曼光谱图只需要1秒即可完成。
综上所述，本发明与现有技术相比，具有如下技术优点：
(1)本发明将激发激光集中到极短的瞬间脉冲，采用瞬间脉冲来激发样品，并将样品中被激发出的光谱集中到激发光脉冲之间的时间段，再利用单通道高灵敏光电探测器对时间上展开的光谱实现高效率的直接测量，而不需要再依赖多通道探测设备测量光谱。本发明能够采用更加灵敏的单通道光电探测器，不仅可以高效利用光谱中的所有光子，而且还可以从数量级上提高光谱测量的灵敏度，同时也避免了传统光谱仪中必须使用CCD这样的多像元、多通道、低灵敏度、低效的探测器对光谱进行测量的问题。同时，脉冲激光的激发和该脉冲之后的探测时间为一个周期，每个周期内所采集的光谱完全相同，光谱中的光子可以用于重复信号的积分，激发光和产生的光谱光子，两者完全被利用于光谱测量，没有浪费；从而提高了光子利用率，提高光谱测量效率。
(2)本发明提供的光谱测量装置结构更加简单，无需高精度的光栅，光谱分辨率由时间延迟色散的分辨率决定；减少各种滤光片的使用，因为激发光和光谱在时间上是分开探测的，不会在不同波长间存在串扰；且使用单通道探头就可实现全光谱测量，不需要分段测量或者移动光栅；激发波长可以随意调节；可以通过双波长消除本底荧光的干扰，尤其适合在拉曼光谱以及CARS光谱中进行应用。
(3)本发明可以测量低波数拉曼光谱。因为传统基于空间色散的拉曼 光谱仪需要应用长通边界滤光片压制激发激光在光谱仪内的散射，导致靠近激发光波长的光谱被截止了；因此低波数的拉曼光谱(<500cm -1)通常无法测量。应用本发明的延时色散光谱的方法可以避开滤光片的使用，从而让低波数的拉曼光谱得到有效采集。
(4)本发明对于由非线性效应产生的光谱更加有利。光学非线性效应产生的光谱通常需要皮秒或飞秒脉冲激光激发。脉冲光的脉宽越短、功率密度越高，非线性光谱的信号越强。同时光谱会在皮秒或飞秒时间内瞬时产生，利用脉冲之间的时间间隙做延时光谱采集时，光谱会被分的更开，时间上重叠部分会更少，光谱分辨率将得到极大提高。
(5)本发明中的单通道高速高灵敏光电探测器包括但不限于光电倍增管(PMT)、硅基光电倍增管(SiPM)、光子计数器、单光子雪崩二极管(SPAD)、调制解调光电放大探测器等实现光谱的探测。因为这些探测器在光电放大增益上和压制信号噪声方面都比传统光谱仪像机有数量级上的提高，因此本发明极大的提高了光谱测量的灵敏度和光子利用率。
(6)本发明在延时色散模块中使用光栅或其它空间色散元件将光谱中不同波长的光子依次衍射到不同的角度出射，然后再进入两片几乎平行的反射镜进行多次反射再原路返回光栅；可以极大的简化光谱仪的结构，去除了传统光谱仪中所需的各种滤光片。且在另外两种类型的延时色散模块中使用色散光纤或者波分器，可以进一步完全去除光栅，结构更加简单，可以进一步小型化，且可靠性更高。
(7)本发明结合各种特异性拉曼标记探针，基因靶向到生物体的多种特定蛋白质或者特定RNA，实现蛋白质组和RNA转录组的识别和多色高速成像。
【附图说明】
图1是基于脉冲激发的延时色散光谱测量原理图，其中，(a)为根据现有的基于空间色散光谱方法获得的光谱示意图，(b)为根据本发明提供 的延时色散光谱方法获得的光谱示意图；
图2是现有技术提供的拉曼过程和CARS过程的能级跃迁图；
图3是本发明提供的脉冲型延时色散光谱测量方法的实现流程图；
图4是本发明提供的脉冲型延时色散光谱测量装置的结构原理框图；
图5是本发明实施例提供的基于脉冲激发的延时色散自发拉曼光谱成像装置的结构示意图；
图6是本发明实施例提供的延时色散CARS光谱成像装置的结构示意图；
图7是本发明提供的相反时间色散的宽光谱泵浦和斯托克斯光产生CARS的原理图；
图8是本发明实施例提供的三种延时色散模块的结构示意图，其中(a)为延时色散原理的示意图；(b)第一实施例提供的基于光纤延时色散原理实现的延时色散模块的结构示意图；(c)第二实施例提供的基于反射镜延时色散原理实现的延时色散模块的结构示意图；(d)第三实施例提供的基于波分器延时色散原理实现的延时色散模块的结构示意图。
【具体实施方式】
为了使本发明的目的、技术方案及优点更加清楚明白，以下结合附图及实施例，对本发明进行进一步详细说明。应当理解，此处所描述的具体实施例仅仅用以解释本发明，并不用于限定本发明。
本发明提出了一种基于脉冲激发和时间延迟色散的新型光谱测量与光谱成像的方法和系统，旨在解决现有光谱测量与光谱成像技术中由于光谱仪的探测灵敏度不足导致光谱测量效率低，光谱成像速度慢的问题。本发明巧妙的结合了短脉冲激光、延时色散光谱，高速高灵敏探测器，突破传统空间色散光谱仪的概念，在时间维度上实现延时色散光谱仪，仅仅利用一个通道的高灵敏探头便可完成光谱的测量。延时色散光谱测量的方法摆脱了现有空间色散光谱仪只能采用低效线阵相机或者低灵敏度光电转换阵 列获取光谱的困境，可以利用高增益或者单光子探测器大幅提高测量灵敏度。该方法在步骤上先利用短脉冲光激发样品产生瞬时光谱，再将光谱中光子按波长或其它光谱特性在时间上分开，最后利用单高速高灵敏光电探测器的光子探测优势，探测光谱中随时间而来的不同光子。
如图3所示，本发明提供了一种脉冲型延时色散光谱测量方法，包括下述步骤：
S1：通过脉冲激光激发待测样品并产生瞬时光谱；
S2：将瞬时光谱中的不同光子在时间上进行延时处理并依次分开；
S3：采用高速高灵敏光电探测器探测光谱中随时间而来的光子。
在步骤S1中，短脉冲激光(具体是指脉冲宽度时间为1微秒～1阿秒的脉冲激光)与样品相互作用时产生瞬时的光谱(1微秒～1阿秒)，该光谱包括荧光光谱(用于分析分子原子和分子的电子态能级跃迁)、拉曼散射光谱(用于分析分子的振动态能级跃迁)、吸收光谱(用于分析分子的振动态能级跃迁)、或者其它光谱(包括但不限于转动光谱、红外吸收光谱等，可以帮助分析原子和分子的化学和物理特性)。
在步骤S2中，将瞬时光谱中不同光子按波长或其它光谱特性(能量、频率、强度、偏振、波前、相位、波矢方向)的顺序在时间上前后延时并分散开，直到下一个激光脉冲到来。光谱利用了激发脉冲之间的时间间隙做足够的时间上的展开，在不同的时刻，不同波长或其它光谱特性的光子到达探测器，同时保证高速光电探测器能够按时间依次探测不同波长或光谱特征的光子，再根据到达时间反推出光谱。
在步骤S3中，可以使用单通道高速高灵敏度的光电探测器探测按不同延时到来的不同波长或光谱特性的光子。单通道高速高灵敏光电探测器包括但不限于光电倍增管(PMT)、硅基光电倍增管(SiPM)、光子计数器、单光子雪崩二极管(SPAD)、调制解调光电放大探测器等。
在本发明实施例中，单次脉冲激发和该脉冲之后的探测时间为一个周 期，每个周期内所采集的光谱完全相同，可以用于重复信号的积分。脉冲激光器的重复频率就是每秒光谱探测的次数，脉冲与信号采集需要精确同步，脉冲之后的拉曼光谱才能准确叠加和积分。脉冲激光器实时输出激光脉冲的触发信号给延时光谱采集卡，同步激光脉冲和光谱采集。
本发明避开了传统光谱仪中必须使用低灵敏度相机中的像元阵列作为光谱探测器的弊端，而在时间域开辟了延时色散光谱方法，采用单通道高灵敏探测器分时探测光谱。因为本发明采用了瞬时脉冲激光激发光谱，通过延时色散后，不同波长分时而来。因此本发明中，分时探测光谱中不同波长的方法，并不会像通常分时复用方法中出现占空比小，浪费光子问题，反而规避了单通道探头只探测一个波长，浪费其余波长的问题。反而利用单探头探测器(通常具有单光子探测能力)的优势大大提高了光谱测量的灵敏度，高效利用了光谱中微弱的光子，在量级上提高了采谱速度，实现高速光谱成像。
如图4所示，本发明还提供了一种脉冲型延时色散光谱测量装置，包括：脉冲激光发生模块、延时色散模块和光电探测模块，激光发生模块用于产生波长为λl的短脉冲激光，并将短脉冲激光照射至待测样品上用于激发待测样品产生瞬时光谱；延时色散模块用于将瞬时光谱中的不同光子在时间上进行延时处理并依次分开；光电探测模块用于探测光谱中随时间而来的光子。
在本发明实施例中，延时色散模块将瞬时光谱中不同光子按波长或其它光谱特性(能量、频率、强度、偏振、波前、相位、波矢方向)的顺序在时间上前后延时并分散开，直到下一个激光脉冲到来。不同波长或光谱特性的光子在延时色散单元中产生的延时与其波长一一对应。
作为本发明的一个实施例，延时色散模块中可使用色散光纤对光谱中不同波长的光子进行延时，在光纤的出射口保证高速探测器按时间依次探测不同波长的光子。
作为本发明的另一个实施例，延时色散模块中可使用光栅将光谱中不同波长的光子依次衍射到不同的角度出射，然后再进入两片几乎平行的反射镜进行多次反射再原路返回光栅。因为光谱中不同波长的光子在反射镜之间来回的路程差不同，因此不同波长的光子产生不同的时间延迟。保证高速探测器在光的出射口按时间依次探测不同波长的光子。
作为本发明的另一个实施例，延时色散模块中可使用波分器将光谱中不同波长的光子分发到不同的光纤波导中，由于光纤波导的长度不同，导致不同波长的光子的传播距离不同，最后将不同路的光经过光复用器进行合并输出，实现光谱中不同波长的光子具有不同的延时，保证高速探测器按时间依次探测不同波长的光子。
在本发明实施例中，激光发生模块产生的短脉冲激光可以激发出样品中的瞬时荧光光谱；延时色散模块将瞬时荧光光谱中不同光子按波长顺序在时间上前后延时并分散开，直到下一个激光脉冲到来。荧光光谱利用了激发脉冲之间的时间间隙做足够的时间上的展开，保证高速光电探测器能够按时间依次探测不同波长的光子。
作为本发明的一个实施例，激光发生模块产生的短脉冲激光可以激发出样品中的瞬时自发拉曼散射光谱。延时色散模块将瞬时自发拉曼散射光谱中不同光子按波长顺序在时间上前后延时并分散开，直到下一个激光脉冲到来。拉曼光谱利用了激发脉冲之间的时间间隙做足够的时间上的展开，保证高速光电探测器能够按时间依次探测不同波长的光子。
本发明在测量装置上的优势是不仅在量级上实现了光谱探测灵敏度的提高，同时简化了光谱仪的结构，整机可进一步小型化，提高可搬运能力，降低制造成本。
由于现有技术中的光谱测量效率太低，速度太慢，如果采用现有技术中的测量方式实现光谱成像，1000ⅹ1000个像素的光谱，就要在短时间里采集1百万个光谱，需要几天时间，完全不现实。然而，由于本发明可以在 量级上提高光谱测量的效率和速度，因此能够进一步实现光谱成像了。
本发明还提供了一种基于脉冲型延时色散光谱测量的光谱成像方法，该成像方法之所以能够解决现有技术中由于光谱测量效率低导致光谱成像速度慢的技术问题，正因为采用了前文中描述的光谱测量方法，正式由于光谱测量方法能够提高光谱测量效率，使得本发明中的成像方法具有成像速度快的优点。例如，成像方法可以是通过移动样品台，或者用激光扫描样品进行成像等常规技术手段来实现。至于成像方法的具体实现步骤是现有技术，在此不再赘述。
在本发明中，样品的在三维空间上的光谱成像方式可以应用但不限于三维激光扫描或三维样品平移台。
本发明还可以结合表面增强拉曼(Surface Enhanced Raman Scattering,SERS)，实现更加灵敏的拉曼光谱探测和成像，以及实现拉曼光谱与背景光谱的分离。
本发明实施例提供的光谱成像方法还可以结合特异性拉曼标签，包括但不限于，基因改造生成的类似于GFP蛋白结构中HBI的分子；具有碳碳双键或者碳碳三键或者其它具有π键的拉曼信号较强的染料分子，如罗丹明800或其类似分子等；基因靶向到生物体的多种特定蛋白质或者特定RNA，实现蛋白质组和RNA转录组的识别和多色成像。
本发明实施例中，泵浦光(Pump)和斯托克斯光(Stokes)为短脉冲激光，在空间和时间上重叠，由于非线性效应激发出样品中的瞬时反斯托克斯拉曼光子(Coherent Anti-Strokes Raman Scattering，CARS)光谱。延时色散模块将瞬时CARS光谱中不同光子按波长顺序在时间上前后延时并分散开，直到下一个激光脉冲到来。CARS光谱利用了激发脉冲之间的时间间隙做足够的时间上的展开，保证高速光电探测器能够按时间依次探测不同波长的光子。
其中，泵浦光(Pump)宽线宽飞秒激光，或者超连续谱飞秒激光；斯 托克斯光(Stokes)为窄带脉冲激光，这样可以实现CARS光谱。
另外一个选择，泵浦光(Pump)和斯托克斯光(Stokes)都为宽线宽飞秒激光，或者两者都是超连续谱飞秒激光；但两者的时间色散正好相反，同样可以产生CARS光谱。该方法因为采用了两束飞秒光，光子没有浪费，CARS光谱产生的效率更高。
为了更进一步的说明本发明实施例提供的脉冲型延时色散光谱测量方法、装置及光谱成像方法，现结合具体实例并参照说明书附图详述如下：
实例1：基于脉冲激发的延时色散自发拉曼光谱测量与光谱成像实例。
图1为基于脉冲激发的延时色散光谱测量的原理图，其中(a)为根据现有的基于空间色散光谱方法获得的光谱示意图，(b)为根据本发明提供的延时色散光谱方法获得的光谱示意图；从图(a)中可以看出：光谱在空间上色散分开，应用光谱仪的相机阵列的众多像元在探测光谱的不同成分，但阵列像元的灵敏度普遍很低，光子利用率较差。从图(b)中可以看出：光谱在时间维度上分开，虚线为激发光短脉冲，实线为在时间上延时后的光谱，高速高灵敏度的单通道光电探头可以按时间顺序探测延时到来的光谱成分。延时色散光谱在时间上可以重复不断的进行测量和积分。
本发明中，短脉冲激光的重复序列，激发样品产生瞬时光谱，再利用延时色散模块将光谱中不同波长的光子在激发脉冲之间的时间间隙上按波长顺序延时并依次分开，最后采用高速高灵敏光电探测器按时间依次探测不同波长的光子，再根据到达时间反推出光谱。单次脉冲激发和该脉冲之后的探测时间为一个周期，每个周期内所采集的光谱完全相同，可以用于重复信号的积分。脉冲激光器的重复频率就是每秒光谱探测的次数，脉冲与信号采集需要精确同步，脉冲之后的光谱才能准确叠加和积分。
图2示出了现有技术提供的拉曼过程和CARS过程的能级跃迁图，其中拉曼过程为：样品中的分子受到激光的激发产生拉曼散射光子；CARS过程为：样品中的分子受到相位锁定的脉冲激光，泵浦Pump光与斯托克斯 Stokes光(两种光子的能量差等于分子基态的跃迁能量)，产生反斯托克斯Anti-Stokes光子。在拉曼光谱的测量中，激光被样品散射后，如图2所示，散射光子的能量与激发光的光子能量相差一个分子的振动能级差。因此通过探测拉曼散射光子的光谱就可以识别样品中的分子种类和相对浓度。
本发明实施例还提供了一种基于脉冲激发的延时色散拉曼光谱测量与光谱成像装置，参考图5，该装置中各种系统参数可根据实际情况选定。
本发明实施例提供的脉冲型延时色散光谱成像装置包括：脉冲激光发生模块1、第一反射模块102、激光扫描模块103、物镜聚焦模块104、样品台105、光子收集模块106、延时色散模块2、光电探测模块3和采集模块4；脉冲激光发生模块1用于产生脉冲激光；第一反射模块102用于将脉冲激光反射至激光扫描模块103；激光扫描模块103用于在空间上扫描激光并实现成像；物镜聚焦模块104用于聚焦激光至样品上；样品台105用于放置样品；光子收集模块106用于收集样品产生的瞬时光谱并对其准直后输出；延时色散模块2用于将瞬时光谱中的不同光子在时间上进行延时处理并根据时序先后依次分开；光电探测模块3用于探测光谱中随时间而来的光子；采集模块4用对探测的光子进行采集。
该成像装置的工作过程如下：脉冲激光经过第一反射模块102进入成像显微镜的三维或二维激光扫描模块103以及物镜聚焦模块104，完成激光对样品的扫描成像。激光扫描系统可以是扫描振镜与其它激光扫描方式的组合。三维位移样品台105也同样可以完成样品的三维扫描。激光与样品相互作用产生的拉曼光子通过光子收集模块106后进入延时色散模块2(具体细节参见实例3和图8)。单通道高速高灵敏探测器进行光电探测，采集模块4将光谱传输到电脑或其它数据终端。
作为本发明的一个实施例，光子收集模块106可以为前向探测光子收集模块，也可以为后向探测光子收集模块(图5中虚线部分)；前向探测光子收集模块用于在透过样品的方向收集样品产生的瞬时光谱并输出；后 向探测光子收集模块用于在激发光反方向收集样品产生的瞬时光谱并输出。
具体地，脉冲激光自发拉曼光源可发出重复频率为1MHz的1ns脉宽的激光脉冲，激光的平均功率为100mW～200mW，波长为532nm。脉冲激光经过第一反射模块102进入成像显微镜的三维或二维激光扫描模块103以及物镜聚焦模块104，完成激光对样品的扫描成像。激光扫描系统可以是扫描振镜与其它激光扫描方式的组合。三维位移样品台105也同样可以完成样品的三维扫描。激光与样品相互作用产生的拉曼光子通过光子收集模块106，可以为共聚焦收集系统，也可以为光纤收集系统，进入核心部件延时色散模块2(具体细节参见实例3和图8)，完成拉曼光子中不同波长成分的延时色散。也可以通过后向探测(图5中虚线部分)进入延时色散模块。因为激光脉冲的重复频率为1MHz，每个脉冲之间的时间间隙为1000ns。延时色散模块将收集到的拉曼的斯托克斯光子，波长通常为533nm～680nm，大概覆盖拉曼位移0～4000cm -1，在1000ns里延时分开成1000段。延时之后，不同波长的光子按波长和时间先后进入单通道高速高灵敏探测器进行光电探测。探测器的采用率为1GHz，每1ns采样一个波长。因此从每一个脉冲激发起的每1微秒都可采集一幅拉曼光谱。通过精确的时序控制，1秒钟可采集100万个拉曼光谱进行积分叠加，得到信噪比更高的拉曼光谱。最后，通过采集模块4将光谱传输到电脑或其它数据终端。同样的，可以等待每一个脉冲或者多个脉冲之后的光谱采集完成，将激光移动到新的位置，采集新的拉曼光谱，那就可以实现拉曼光谱的样品成像。本发明仅仅单通道高灵敏的探头实现了光谱测量和采集，拉曼光子的利用效率和拉曼光谱成像速度都可极大的提高。
该实例同样可以应用于荧光光谱的测量与荧光光谱成像。
实例2：延时色散CARS光谱测量与CARS光谱成像实例。
当激发激光是两束同步且相位锁定的脉冲光源ω P、ω s，其中ω P和ω s， 通常为泵浦光和斯托克斯光；当ω P和ω s光子之间的能量差与分子振动能级差一致时，就会产生反斯托克斯光子ω CARS，如图2所示。
本发明实施例提供了一种延时色散CARS光谱测量和CARS光谱成像的方法，请见参考图6，且各种系统参数可根据实际情况选定。
本发明实施例提供的脉冲型延时色散光谱成像装置包括：双通道脉冲激光发生模块200、第一功率调节模块201、第二功率调节模块202、光调制模块203、时间延迟器204、第二反射模块206、二向色镜模块205、激光扫描模块103、物镜聚焦模块104、样品台105、光子收集模块106、延时色散模块2、光电探测模块3和采集模块4；双通道脉冲激光发生模块200用于产生泵浦光和斯托克斯光两路脉冲激光；第一功率调节模块201用于调节斯托克斯光的功率大小；第二功率调节模块202用于调节泵浦光的功率大小；光调制模块203用于对斯托克斯光的功率或者相位或者偏振或者延时进行调制；时间延迟器204用于在时间上对泵浦光和斯托克斯光的脉冲进行配准；第二反射模块206用于对泵浦光进行反射至二向色镜模块205；二向色镜模块205用于对斯托克斯光进行反射，并在空间上使得泵浦光和斯托克斯光重合；激光扫描模块103用于在空间上扫描激光并实现成像；物镜聚焦模块104用于聚焦激光至样品上；样品台105用于放置样品；光子收集模块106用于收集样品产生的瞬时光谱并对其准直后输出；延时色散模块2用于将瞬时光谱中的不同光子在时间上进行延时处理并根据时序先后依次分开；光电探测模块3用于探测光谱中随时间而来的光子；采集模块4用对探测的光子进行采集。
该成像装置的工作过程如下：双通道脉冲CARS激光光源发出两束飞秒激光：泵浦光ω Pump和斯托克斯光ω Stokes，斯托克斯脉冲激光经过第一功率调节模块201后被光调制模块203调制。时间延迟器204可以调节斯托克斯光的时间延迟，二向色镜模块205将斯托克斯光与泵浦光在空间上完全合束，时间延迟器204和二向色镜模块205两者用于保证斯托克斯光与 泵浦光脉冲在时间上和空间上完全重合。两束飞秒光进入成像显微镜的三维或二维激光扫描模块103以及物镜聚焦模块104，完成激光对样品的扫描成像。三维位移样品台105也同样可以完成样品的三维扫描。激光与样品相互作用产生的CARS光子通过光子收集模块106后进入延时色散模块2。核心部件延时色散模块2(具体细节参见实例3和图8)，完成拉曼光子中不同波长成分的延时色散。延时之后的不同波长的光子按波长和时间先后进入单通道高速高灵敏探测器3进行光电探测。最后，通过采集模块4将光谱传输到电脑或其它数据终端。
作为本发明的一个实施例，光子收集模块106可以为前向探测光子收集模块，也可以为后向探测光子收集模块(图6中虚线部分)，前向探测光子收集模块用于在透过样品的方向收集样品产生的瞬时光谱并输出；后向探测光子收集模块用于在激发光反方向收集样品产生的瞬时光谱并输出。
具体地，双通道脉冲CARS激光光源可发出两束重复频率为1MHz相位锁定的飞秒激光，泵浦光ω Pump和斯托克斯光ω Stokes，激光的平均功率为100mW～1000mW，波长分别为超连续谱800nm～960nm和窄带1040nm。斯托克斯脉冲激光经过第一功率调节模块201后被光调制模块203调制。时间延迟器204可以调节斯托克斯光的时间延迟，二向色镜模块205将斯托克斯光与泵浦光在空间上完全合束，时间延迟器204和二向色镜模块205两者用于保证斯托克斯光与泵浦光脉冲在时间上和空间上完全重合。两束飞秒激光进入成像显微镜的三维或二维激光扫描模块103以及物镜聚焦模块104，完成激光对样品的扫描成像。激光扫描系统可以是扫描振镜与其它激光扫描方式的组合。三维位移样品台105也同样可以完成样品的三维扫描。激光与样品相互作用产生的CARS光子通过光子收集模块106进入延时色散模块2(具体细节参见实例3和图8)，完成拉曼光子中不同波长成分的延时色散。也可以通过后向探测(虚线部分)进入延时色散模块。因 为激光脉冲的重复频率为1MHz，每个脉冲之间的时间间隙为1000ns。延时色散模块将收集到的拉曼的斯托克斯光子，波长通常为650nm～891nm，大概覆盖拉曼位移2885cm -1～801cm -1，在1000ns里延时分开成1000段。延时之后，不同波长的光子按波长和时间先后进入单通道高速高灵敏探测器进行光电探测。探测器的采用率为1GHz，每1ns采样一个波长。因此从每一个脉冲激发起的每1微秒都可采集一幅拉曼光谱。通过精确的时序控制，1秒钟可采集100万个拉曼光谱进行积分叠加，得到信噪比更高的拉曼光谱。最后，通过采集模块4将光谱传输到电脑或其它数据终端。同样的，可以等待每一个脉冲或者多个脉冲之后的光谱采集完成，将激光移动到新的位置，采集新的拉曼光谱，那就可以实现拉曼光谱的样品成像。本发明仅仅单通道高灵敏的探头实现了CARS光谱测量和采集，CARS光子的利用效率和拉曼光谱成像速度都可极大的提高。
另外，泵浦光(Pump)和斯托克斯光(Stokes)也可以都为宽线宽飞秒激光(10fs)，或者两者都是超连续谱飞秒激光，但两者的中心波长差覆盖分子的振动谱，且时间色散正好相反，如图7所示，同样可以产生CARS光谱。泵浦光和斯托克斯光都为宽线宽脉冲激光，两者的光子能量差覆盖分子的振动谱ω i，且时间色散正好相反，保证在一个时刻只要一种分子振动被激发产生CARS光子。
实例3：三种延时色散模块的具体结构实例。
本发明实施例提供了三种产生延时色散光谱的实例。请见参考图8，且各种系统参数和方法可根据实际情况选定。其原理如图8(a)，脉冲激光产生的瞬时光谱波长在时间上是叠加的，利用延时色散的方法，可以将光谱里的光子在时间和波长上分开，并进行探测。
图8(b)示出了第一实施例提供的基于光纤延时色散原理实现的延时色散模块的结构，光纤延时色散模块包括：色散光纤21，其一端连接光子收集模块106，另一端连接光电探测模块3；光子收集模块106收集并耦合 所有光子进入色散光纤21。当不同波长的光子在光纤出口随时间依次出射时，高速高灵敏光电探测模块3对其进行光电探测产生光谱。该方法简单直接且不需要光栅或其它色散元件。
图8(c)示出了第二实施例提供的基于反射镜延时色散原理实现的延时色散模块的结构，反射镜延时色散模块包括：依次摆放的分光元件22、共轭模块23、分束模块24和延时反射模块25；分光元件22用于对光子收集模块106收集后的光子进行分光，使得光谱中不同波长的光子依次衍射到不同的角度后出射；共轭模块23用于将出射光导入延时反射模块25中；延时反射模块25用于对导入的光进行多次反射后再原路返回，使得光谱中不同波长的光子在反射来回的路程差不同实现了不同波长的光子产生不同的时间延迟；分束模块24用于将返回的延时后的光子转移至光电探测模块3。
其中，分光元件22与共轭模块23之间的距离为共轭模块23的焦距，共轭模块23中包括两个透镜，且两个透镜之间的距离为两个透镜的焦距之和。分束模块24设置在分光元件22和延时反射模块25之间的任意位置。
在本发明实施例中，分光元件22可以采用光栅、棱镜等空间色散元件。现以光栅为例详述该反射镜延时色散模块的工作过程如下：
光子收集模块106将光子收集并准直后，所有光子进入光栅或其它空间色散元件进行分光，将光谱中不同波长的光子依次衍射到不同的角度出射。然后通过两个透镜组成的共轭模块23，将光导入两片几乎平行的延时反射模块25进行多次反射再原路返回。延时反射模块25中两块反射板之间的夹角为0.001度～1度；优选地，角度为0.01度。
因为光谱中不同波长的光子在反射镜之间来回的路程差不同，因此不同波长的光子产生不同的时间延迟。延时后的光子返回时被分束模块24转移到光电探测模块3进行探测。保证高速探测器在光的出射口按时间依次探测不同波长的光子。该具体实例可以通过不同光子的光程差实现更大的 延时。
图8(d)示出了第三实施例提供的基于波分器延时色散原理实现的延时色散模块的结构，波分器延时色散模块包括：依次设置的光波分器26、光纤波导27和光复用器28；光子收集模块106收集并耦合所有光子进入光波分器26。光波分器26将光谱中不同波长的光子分发到不同长度的光纤波导27中；由于光纤波导的长度不同，导致不同波长的光子的传播距离不同，最后将不同路的光经过光复用器28进行合并输出给光电探测模块3。
其中，光波分器26可以将不同波长的光子分成100～1000路通道。相应地，光纤波导27中可以设置100～1000根长度不同的光纤波导。
本发明中的光纤延时色散模块的结构简单，需要的元件少，可最大可能的进行仪器小型化；反射镜延时色散模块和波分器延时色散模块可提供更多的色散延时，有利于光谱分辨率的提高。
另外，在本发明中，所有具体实例中的单通道高速高灵敏光电探测模块3包括但不限于光电倍增管(PMT)、硅基光电倍增管(SiPM)、光子计数器、单光子雪崩二极管(SPAD)、调制解调光电放大探测器等。单通道探测器比多通道探测器具有更高的光电灵敏度和数据传输速度，也可以有更大的探测面积，提高光子的探测效率。
本领域的技术人员容易理解，以上所述仅为本发明的较佳实施例而已，并不用以限制本发明，凡在本发明的精神和原则之内所作的任何修改、等同替换和改进等，均应包含在本发明的保护范围之内。

Claims (12)

  1. 一种脉冲型延时色散光谱测量方法，其特征在于，包括下述步骤：
    S1：通过脉冲激光激发待测样品并产生瞬时光谱；
    S2：将瞬时光谱中的不同光子在时间上进行延时处理并根据时序先后依次分开；
    S3：采用高速高灵敏光电探测器探测光谱中随时间而来的光子。
  2. 如权利要求1所述的光谱测量方法，其特征在于，在步骤S2中，将瞬时光谱中不同光子按波长、频率、相位、能量、偏振、波矢方向或强度在时间上进行延迟并分散开。
  3. 如权利要求1或2所述的光谱测量方法，其特征在于，在步骤S3中，所述高速高灵敏光电探测器采用单通道探测元件，包括光电倍增管、硅基光电倍增管、光子计数器、单光子雪崩二极管或调制解调光电放大探测器。
  4. 一种脉冲型延时色散光谱测量装置，其特征在于，包括脉冲激光发生模块(1)、延时色散模块(2)和光电探测模块(3)；
    所述脉冲激光发生模块(1)用于产生脉冲激光，并将所述脉冲激光入射至待测样品上激发样品产生瞬时光谱；
    所述延时色散模块(2)用于将瞬时光谱中的不同光子在时间上进行延时处理并根据时序先后依次分开；
    所述光电探测模块(3)用于探测光谱中随时间而来的光子。
  5. 一种基于权利要求1所述的光谱测量方法实现的光谱成像方法。
  6. 一种脉冲型延时色散光谱成像装置，其特征在于，包括：脉冲激光发生模块(1)、第一反射模块(102)、激光扫描模块(103)、物镜聚焦模块(104)、样品台(105)、光子收集模块(106)、延时色散模块(2)、光电探测模块(3)和采集模块(4)；
    所述脉冲激光发生模块(1)用于产生脉冲激光；
    所述第一反射模块(102)用于将所述脉冲激光反射至所述激光扫描模块(103)；
    所述激光扫描模块(103)用于在空间上扫描激光并实现成像；
    所述物镜聚焦模块(104)用于聚焦激光至样品上；
    所述样品台(105)用于放置样品并移动样品完成成像；
    所述光子收集模块(106)用于收集样品产生的瞬时光谱并输出；
    所述延时色散模块(2)用于将瞬时光谱中的不同光子在时间上进行延时处理并根据时序先后依次分开；
    所述光电探测模块(3)用于探测光谱中随时间而来的光子；
    所述采集模块(4)用对探测的光子进行采集。
  7. 一种脉冲型延时色散光谱成像装置，其特征在于，包括：双通道脉冲激光发生模块(200)、第一功率调节模块(201)、第二功率调节模块(202)、光调制模块(203)、时间延迟器(204)、第二反射模块(206)、二向色镜模块(205)、激光扫描模块(103)、物镜聚焦模块(104)、样品台(105)、光子收集模块(106)、延时色散模块(2)、光电探测模块(3)和采集模块(4)；
    所述双通道脉冲激光发生模块(200)用于产生泵浦光和斯托克斯光两路脉冲激光；
    所述第一功率调节模块(201)用于调节斯托克斯光的功率大小；
    所述第二功率调节模块(202)用于调节泵浦光的功率大小；
    所述光调制模块(203)用于对斯托克斯光的功率或者相位或者偏振或者延时进行调制；
    所述时间延迟器(204)用于在时间上对所述泵浦光和所述斯托克斯光的脉冲进行配准使得泵浦光与斯托克斯光脉冲在时间上重合；
    所述第二反射模块(206)用于对所述泵浦光进行反射至所述二向色镜 模块(205)；
    所述二向色镜模块(205)用于对所述斯托克斯光进行反射，并在空间上使得所述泵浦光和所述斯托克斯光重合；
    所述激光扫描模块(103)用于在空间上扫描激光并实现成像；
    所述物镜聚焦模块(104)用于聚焦激光至样品上；
    所述样品台(105)用于放置样品并移动样品完成成像；
    所述光子收集模块(106)用于收集样品产生的瞬时光谱并输出；
    所述延时色散模块(2)用于将瞬时光谱中的不同光子在时间上进行延时处理并根据时序先后依次分开；
    所述光电探测模块(3)用于探测光谱中随时间而来的光子；
    所述采集模块(4)用对探测的光子进行采集。
  8. 如权利要求6或7所述的光谱成像装置，其特征在于，所述光子收集模块(106)为前向探测光子收集模块或后向探测光子收集模块；
    所述前向探测光子收集模块用于在透过样品的方向收集样品产生的瞬时光谱并输出；
    所述后向探测光子收集模块用于在激发光反方向收集样品产生的瞬时光谱并输出。
  9. 如权利要求7所述的光谱成像装置，其特征在于，当所述泵浦光和所述斯托克斯光均为宽线宽飞秒激光或者超连续谱飞秒激光，且所述泵浦光和所述斯托克斯光的中心波长差覆盖分子的振动谱，且所述泵浦光和所述斯托克斯光的时间色散正好相反时产生CARS光谱。
  10. 如权利要求4、6-9任一项所述的装置，其特征在于，所述延时色散模块(2)包括：色散光纤(21)，其一端连接所述光子收集模块(106)，另一端连接所述光电探测模块(3)；当不同波长的光子在所述色散光纤(21)出口随时间依次出射时，所述光电探测模块(3)对其进行光电探测并产生光谱。
  11. 如权利要求4、6-9任一项所述的装置，其特征在于，所述延时色散模块(2)包括：分光元件(22)、共轭模块(23)、分束模块(24)和延时反射模块(25)；
    所述分光元件(22)用于对所述光子收集模块(106)收集后的光子进行分光，使得光谱中不同波长的光子依次衍射到不同的角度后出射；
    所述共轭模块(23)用于将出射光导入所述延时反射模块(25)中；
    所述延时反射模块(25)用于对导入的光进行多次反射后再原路返回，使得光谱中不同波长的光子在反射来回的路程差不同实现了不同波长的光子产生不同的时间延迟；
    所述分束模块(24)用于将返回的延时后的光子转移至所述光电探测模块(3)。
  12. 如权利要求4、6-9任一项所述的装置，其特征在于，所述延时色散模块(2)包括：光波分器(26)、光纤波导(27)和光复用器(28)；
    所述光波分器(26)用于将所述光子收集模块(106)收集后的光子分发至不同长度的所述光纤波导(27)中；
    不同长度的光纤波导(27)使得不同波长的光子的传播距离不同，实现了不同波长的光子产生不同的时间延迟；
    所述光复用器(28)用于将时间延迟后的多路光子进行合并并输出给所述光电探测模块(3)。
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