Study On Broadband Coherent Anti-stokes Raman Scattering Spectroscopy And Microscopy

Coherent anti-Stokes Raman scattering?CARS?can recognize and locate specific molecules through their intrinsic vibrational spectrum,eradicating necessity of exogenous labels.Furthermore,it demonstrates such advantages as high sensitivity,high speed,high temporal and spatial resolution,enabling noninvasive microscopic spectral imaging of organism tissue and biological macromolecules of the living cell in situ,in vivo and in real time.CARS has found many applications in biomedical researches of lipids,protein,nucleic acids and so on.Traditional CARS can only image only single resonant molecular bond,due to the limitation of pump and Stokes spectral bandwidth.Obviously,it is not enough to acquire accurate recognition and adequate mapping of the unknown biomolecules or their complexes.To obtain the whole Raman spectrum for recognizing different types of macromolecules,it is necessary to tune the wavelength of pump or Stokes frequently.For this reason,some researchers proposed broadband CARS to probe several vibrational modes simultaneously by using the narrowband pulse and broadband continuum pulse for accurate molecular recognition.Limited by the temporal and spectral structure of continuum pulses,the present-day broadband CARS often adopts two-color CARS mechanism,in which the narrowband pulse serves as both pump and probe and the continuum pulse works as only Stokes.Therefore,it is quite difficult to suppress the non-resonant background?NRB?by time-resolved detection.In this thesis,we present a three-color ultra-broadband spectroscopy and microscopy,which utilize a femtosecond fiber laser and all-normal dispersion photonic crystal fiber.This method adopts the supercontinuum with good temporal and spectral structure?bandwidth>500 nm and pulse duration<220 fs?generated by injecting femtosecond pulses into a NL-1050-NEG all-normal dispersion photonic crystal fiber as both pump and Stokes.Therefore,the whole CARS spectrum of biomolecule can be acquired simultaneously,and the NRB can also be effectively suppressed by controlling time delay between the continuum pulse and narrowband laser pulse.In this way,the recognition of different macromolecules becomes much more accurate.Furthermore,the time-resolved method can also be used to measure the vibrational dephasing process,which has important application in studies of molecular dynamics and interactions.The main works which this thesis are as follows:1.Procedures for point-by-point scanning control and image reconstruction have been developed.To test reliability of the program,a CARS microscopy setup has been engineered and built basing on the Ti:sapphire femtosecond laser and optical parametric oscillator.The functionality of the constructed system has been demonstrated by obtaining CARS images of polystyrene beads with different diameter.The lateral resolution was also calibrated with a 110 nm diameter polystyrene bead.2.To realize a broadband CARS spectroscopy and microscopy,we numerically simulated the supercontinuum generation in PCFs with two-zero-dispersion wavelength and all-normal dispersion.By analyzing the mechanism and characteristics of supercontinuum generated in this two PCFs,we have found that supercontinuum generated with all-normal dispersion PCF has a relatively high coherence and stability,which is very suitable for time-resolved applications.Therefore,the supercontinuum generated with all-normal dispersion PCF is proposed for the three-color broadband CARS spectroscopy.3.A two-color broadband CARS spectroscopy has been developed and the setup based on the Ti:sapphire femtosecond laser has been built.Analysis of variation of CARS sample signal?from neat dimethylsulphoxide?with different narrowband pulse and supercontinuum pulse power indicates that the mechanism is the two-color CARS process.In this two-color broadband CARS system,we obtained the broadband spectrum and images of polystyrene beads.4.Based on a single femtosecond fiber laser,three-color ultra-broadband CARS spectroscopy has been developed and the corresponding setup has been built.With this system,we have instantaneously obtained the whole CARS spectrum of the sample?neat benzonitrile?.Also,the time-resolved detection has been employed to effectively suppress the non-resonant background.Furthermore,the vibrational dephasing times of every vibrational mode have been measured by controlling the time delay between the supercontinuum and narrowband pulses.The main innovation in this thesis can be summarized as follows:1.Supercontinuum generated in an all-normal dispersion photonic crystal fiber has been applied to the broadband time-resolved CARS spectroscopy and microscopy.The supercontinuum after dispersion compensation has good temporal and spectral structure?bandwidth>500 nm and pulse duration<220 fs?,spectral continuity and uniformity,high stability and energy efficiency.2.We proposed and realized a three-color ultra-broadband CARS spectroscopy,which can acquire the whole CARS spectrum of biological molecules at once.With this method,all the vibrational mode range from 300 cm^-1 to 3600 cm^-1 can be addressed simultaneously and the NRB can further be effectively suppressed with time-resolved detection.Also,the dephasing times of all the vibrational mode can be measured concurrently.