Study On Spectrally Resolved Multifocal Multiphoton Microscopy Imaging System

Fluorescence microscopy has evolved into a powerful tool in biomedicine. Fluorescence inherently is a multi-parameter signal. Fluorescence spectrum measurement can be used to identify different fluorophores. Fluorescence lifetime on the other hand can be used to analyze quantitatively various physiological parameters in the microenvironment of the flurophores. Lifetime measurement is also proved to be an effective tool to identify flurophores with overlapping spectra. Multifocal multiphoton microscopy (MMM) is a unique technique which can acquire three-dimensional fluorescence images by multiphoton excitation with the advantages of high speed, reduced photobleaching and photodamage, enhanced penetration depth and high signal-noise ratio.The goal of this thesis is to combine the prism spectroscopy and MMM, and develop a multiphoton fluorescence microscopy system that is capable of performing fast 3-D spectral measurements of fluorescent samples using two-photon excitation. This will provide an essential tool for many biological studies, such as multi-color imaging, fluorescence resonance energy transfer (FRET) and fluorescence ratio measurements. In this thesis, a Spectrally Resolved Multifocal Multiphoton Microscopy (SR-MMM) system has been developed. In order to obtain spectrally resolved fluorescence intensity image and the spectral profiles of the sample, the thesis deals with the topics related to spectrum measruements. In addition, a spectrally resolved multifocal multiphoton fluorescence lifetime imaging microscopy system has also been built, which has been used for the spectrum and lifetime measurements of biological samples. In summary, this thesis focuses on following issues:1 Fluorescence microscopy techniques and its development that are related to this work has been reviewed.2 An SR-MMM system has been developed to acquire the fast 3-D spectrally resolved fluorescence data of the sample at a higher rate. 3 Data analysis and processing method for SR-MMM system has been studied. Spectral resolution and spatial magnification of the system are calibrated.4 Image reconstruction methods for SR-MMM are investigated so that the intensity image and spectrally resolved intensity image of the sample can be reconstructed and the spectral profiles of the regions of interest in the sample can be obtained.5 A spectrally resolved multifocal multiphoton fluorescence lifetime imaging microscopy system is built and calibrated.In experiment, the SR-MMM system has been used to acquire spectrally resolved multiphoton fluorescence images of various samples, such as prepared plant slide, fixed and live cells. Spectral range and resolution of the system are 450-700nm and <3nm respectively. Experimental results demonstrate the great potentials of this technique in biomedicine.