Two-photon Fluorescence Microscopy Study

The two-photon fluorescence microscopy has been used for three-dimensional imaging by detecting the fluorescence emitting from the specimen when absorbing two photons. It has become an important instrument of real time studying the cellular construct and function in vivo, because it reduces the photodamage, light scattering and increases the resolution compared with one-photon excitation.In this dissertation, the concept of the two-photon fluorescence microscopy is brought out based on the theory of fluorescence and fluorescence microscopy. And the characteristics of two-photon fluorescence imaging are also analyzed compared with one-photon by studying of two-photon imaging theory. What's more, a new optical scanning fiber which can achieve two-dimensional scanning through one-dimensional vibrating of bimorph has been developed. After building the two-photon system, we measure the fluorescent intensity of Rhodamine B with different concentration and improve the system by analyzing the errors in the experiments.This dissertation is composed of five parts. Chapter one explains the background of carrying out the two-photon fluorescence microscopy research, introduces fluorescence and fluorescence microscopy, and the application of two-photon fluorescence microscopy. Chapter two analyzes the two-photon imaging characteristics theoretically and gets the point spread function of two-photon confocal microscopy. Chapter three gives a brief introduction of building the two-photon imaging system and develops a new type of optical scanning fiber. Chapter four contains the experiments carried out and the analysis of the results. Chapter five makes a conclusion of this research and presents the main task in future.