Study Of Scanning Control And Imaging System Of Two Photon Fluorescence Microscopy

Two photon fluorescence microscopy(TPFM) is an effective tool in the study of living tissues and cell structures. The system applies two photon absorption principle to excite the sample fluorescence. The fluorescence excitation probability decreases with the distance away from the focal plane, so that only a very small area of the focal plane is excitated, which has a high imaging resolution and SNR(Signal Noise Ratio). Two photon fluorescence microscopy, in the cell biology, neuroscience, brain science and other biological and medical fields have broad application.In this paper, there are three aspects to discuss TPFM--- control project design(hardware and algorithm), software interface design, and distortion correction algorithm design. The system is demonstrated from the simulation data and experimental results. Principle prototype and its testing are completed. The main work includes the following parts:First, the hardware control system of the TPFM is designed and built. TPFM is a laser scanning microscope system. Scanning control system and signal acquisition are the key technologies. In this paper, the hardware control module is built based on data acquisition card, and the signal is acquired by the photo multiplier tube(PMT).Secondly, the control program and human-computer interaction(HCI) are written. In order to comply with the system, the Lab VIEW is used to build the control software. It can realize the unified control of the scanning module, CCD module, laser module, 3D platform, Objective scanner and other modules. The MATLAB script is embedded in the program to process image signal.As the same time,bovine pulmonary artery endothelial(BPAE)cell was scanned to image,and the 2D image of BPAE was reconstructed.Because TPFM can image deeply at the sample,and obtain thin focal plane signal,it can obtain the different depth information of the sample by using压电驱动器in the Z direction to scan.The images of these layers were reconstructed to obtain 3D image of the sample;Finally, the correction algorithm of the resonant scanning distortion is designed. Due to nonlinearity of resonant scanners’ speed, images are distorted in laser scanning imaging systems based on resonant scanner. An efficient algorithm to correct the distortion is developed, by fitting the distortion curve. The core of correction algorithm is to restore the image using distortion function;At the end of the paper, the research contents are summarized and analyzed, and the prospect of two photon fluorescence microscopy is presented.