High Speed Two-photon Fluorescence Microscopy Based On Galvonometer Scanning With Femtosecond Laser

Two-Photon Laser Scanning Fluorescence Microscopy(2PM)always bears its great significance in the field of biology for its high resolution,deep depth of image and focused bleaching area,by means of which research in biological tissue structure is to be made,especially for noninvasive study.With the development of 2PM,it is gradually applied in a wide range of biological branches.In this thesis,a high speed two-photon microscopy imaging system is developed and studied in theory and experiment to demonstrate the advantages of 2PM and highlight its application value.The Olympus IX71 is applied for microscopic observation and Coherent Micra-5 femtosecond laser as the light source.Galvanometer is introduced as the scanning tool to the sample of Rhodamine B and Caski cells specifically bound with Rhodamine-dyed Phalloidin,improving the imaging speed to 1frame/s.With the sample of Rhodamine B,the influence of 2D imaging quality is studied;the images of different cross-sections is made for 3D reconstruction,which proves the vertical resolution to the order of 3.0?m,then to achieve the three-dimensional microscopic imaging.Furthermore,the image quality of Caski cells bound with Rhodamine-dyed Phalloidin is analyzed and the lateral resolution of cell imaging is proved to reach 1.05?m with the application of 2PM.In theory,the fluorescence intensity is derivated on the ground of the nonlinearity of two-photon absorption.As a result,the fluorescence intensity with the use of 45 fs femtosecond laser is proved to be up to 105 compared with CW laser,thus showing the advantages of femtosecond laser as the light source.Moreover,a quantitative description is given to the advantage of two-photon fluorescence imaging in image depth and resolution;and the saturation effect is analyzed for the feasibility of this two-photon imaging system.In terms of resolution,the space distribution of Gaussian pulse in along and vertical axis is derivated,where a comparison is made between the single and two photon imaging.The theoretical resolution is given in the light of diffraction limit theory and the practical resolution in accurate focused condition is arrived from relay optical path.In software,a program for 2PM is framed with the software of LabVIEW,the core of which is to control the synchronization between the PMT collection and Galvanometer scan.The program is designed in sequential structure inc luding program initialization,output signal waveform generation,synchronous preparation,operation trigger,data acquisition,Z motion and forced stop of task.The design thought and further instruction of the whole frame and the important part is showed in this thesis.It is demonstrated that the program can show 2D image of 2PM,realize automatic cross-section acquisition effectively,and store the acquired data with the name of image start time.In experiment,a high speed two-photon microscopy imaging system is developed,which can both realize the hyper-spectral CARS and Two-photon fluorescence microscopy.It uses pulse shaping to controllably broaden the pulse bandwidth and compensate for spectral phase distortions,thus improving the resolution.By means of galvanometer,the scanning speed is improved to the order of 1 frame/s,whic h compensates the defect of long scanning time of mechanical translation platform,and much damage to cells.With the sample of Rhodamine B,the experiment analyzes the influence of scanning time,voltage value and pulse shaping on imaging capability.By cross section scanning,it is demonstrated that the vertical resolution can reach 3.0?m;and the captured cross section images are 3D-reconstructed to achieve the threedimensional microscopic imaging with Matlab.Furthermore,using Caski cells bound with Rhodamine-dyed Phalloidin as the sample,the fluorescence-image of whic h shows the cell distribution characteristics,and it is demonstrated that the fluorescenceimage of the sample is clearer with the higher power of laser and closer area of focus.The practical resolution is proved to reach 1.05?m by drawing the tendency of pixel gray level at the sharpened line of cell membrane.The microfilament structure of cells is observed in this microscopy system and the ability of the Phalloidin specifical binding to the cell is also verified.