Femtosecond Laser Two-photon Fluorescence Biological Microscopy

As the most important invention in the imaging field, two-photon fluorescencemicroscopy has been widely applied in biological research and other fields. Comparedwith the confocal microscopy, the nonlinear nature of the two-photon fluorescencemakes two-photon fluorescence microscopy have unique advantages, such as enhancedcontrast and resolution and limiting photobleaching to the focal point. In addition, NIRfemtosecond laser excitation pulses have a good ability of penetrating and reducingphotodamage of biological specimens, thus making two-photon fluorescencemicroscopy not only suitable for the long time research but also suitable for great depthimaging of biological tissue In this dissertation, the theory of the two-photonfluorescence microscopy is analyzed, a two-photon fluorescence microscopy system isbuilt and the control software of the system is designed. Some researches on two-photonmicroscopy have been conducted by using this home-built system. What’s more, thesystem has been applied into the biological research.Theoretically, the process of two-photon fluorescence is analyzed, and the timeaveraged two-photon fluorescence intensity at the random time interval T wascalculated. The advantage of ultrashort pulse laser in two-photon fluorescencetwo-photon excitation is also analyzed by formula. The spatial light distribution oftwo-photon microscopy is deduced from equations, and then the advantages of thismicroscopy are obtained by the comparison with one-photon microscopy.In the software design，software module are introduced, including computercontrol module, mechanical scanning module, image acquisition and processing module.Then factors which influence the precision of mechanical scanning location are alsodiscussed。The results show that in order to guarantee the position accurate, set up thesystem for the resolution of1μm. The influence of different pixel interval and singleacquisition or acquisition for many times on imaging results are also discussed. Resultsshow that when selecting multiple acquisition and setting the pixel interval between120-160ms, the imaging results has no distortion and its signal-to-noise ratio is higherIn experiment, two-photon fluorescence spectrum of Rhodamine B solution withdifferent concentrations are measured. The results show that with the concentrationincreases, the centre wavelength of two-photon fluorescence spectrum of Rhodamine Bsolution approaches the maximum fluorescence emission wavelength,610nm.Two-photon fluorescence microscopic imaging and transmission imaging of RhodamineB have been studied based on the system. The imaging result of correspondence hasproved the validity of the imaging system. Through scanning different level of Rhodamine B, the three-dimensional image of the sample has been achieved. At last, weapply the two-photon fluorescence microscopy system into the biological cells.Through applying the two-photon fluorescence microscopy system into the Hela cellscontaminated with DAPI and Rhodamine B, how the z axis position influence theimaging results and the relationship between the incident laser power and the intensityof fluorescenc have been discussed. Furthermore, the dyeing ability for cell nucleus ofDAPI and the dyeing ability for cell of Rhodamine B have been proved.