Study On Laser Microscopy Imaging And CARS Spectrum Detection Technology

With the development of technology, human want to see a more microscopic world. We study not only on the structure of biological tissue or cell morphology, but also know more about the basic composition of biological components-molecules or atoms. Recent years, technology in this field has made great achievements,such as microscopic imaging of biological tissue or cell and molecular detection of substances, among which, the two-photon fluorescence microscopic technology femtosecond laser and the coherent anti-Stokes Raman scattering( CARS) spectroscopic technique, as an important means of microscopic imaging of biological tissue and molecular structure research, have attracting much interests of researchers those days with their unique advantages.The femtosecond laser two-photon fluorescence microscopic technology was considered one of the effective means of biological tissue or cell microscopic imaging, the near-infrared femtosecond laser was chosen for its excitation light source. Because two-photon fluorescence wavelength is away from the excitation wavelength, so the dark field imaging it realized would greatly improves the signal-to-noise ratio, and the near-infrared femtosecond laser has a good ability of penetrating,reducing photo damage of biological specimens and gaining a greater depth of imaging. This reminds one of its important advantages compared with the single photon fluorescence confocal microscopy imaging. In this dissertation, the process of two-photon fluorescence excitation is derived in theory, and then analysis of the spatial distribution of two-photon fluorescence intensity is made, it also contains a comparison to single photon confocal microscopy imaging of its advantages. Through the theoretical research, a set of femtosecond laser two-photon fluorescence microscopy imaging system was built by using a mode-locked titanium sapphire laser under the existing conditions in the laboratory, then the system controlling and scanning part of the software has been wrote, the signal acquisition system was also designed. First of all, two-photon fluorescence spectrum of the rhodamine B solution in different concentrations are measured by the experiment, the result show that with concentration increase, the two-photon fluorescence spectrum generated redshift and the center wavelength will approaches the maximum fluorescence emission wavelength of 610 nm. On this basis, microscopic imaging experiments and analysis of the results of imaging were carried out on rhodamine B samples. By adjusting the z-axis of three-dimensional displacement platform, study of the impact on the imaging results of the distance between the sample and the objective lens focus was made. This also proves the correctness of the theoretical results, which only in the vicinity of the focus can produce the two-photon fluorescence process.This paper also studied the coherent Anti-stokes Raman scattering on the detection of molecular structures. After the theoretical study of third-order nonlinear processes of CARS, time-resolved method is used to suppress non-resonant background noise in the CARS spectrum signal. In order to obtain a complete corresponding CARS spectrum of molecular vibrational modes, femtosecond pulse pump photonic crystal fiber was used to produce super continuous laser spectrum, which achieves the ultra-wideband time-resolved CARS spectrum detection. In experiments, the femtosecond pulse from mode-locked titanium-sapphire laser is split into two parts, one of femtosecond pulse pumped the photonic crystal fiber to generate super continuum laser which would be used as the pump and stokes simultaneously. Other part of the femtosecond pulse is used as the probe light after an appropriate time delay in the process of CARS. The experiment measured the CARS spectrum information of dimethyl sulfoxide at the range of 500 cm-1-3500 cm-1. The effect of temperature on CARS spectra signal of Dimethyl sulfoxide has been dis cussed as well.