Image enhancement with two-photon laser scanning microscopy

Although confocal microscopy provides an efficient means of fluorescence imaging, many obstacles including extensive photobleaching and limited penetration depths limit its application. Emergence of two-photon laser scanning microscopy (TPLSM), with limited excitation volume, successfully overcomes those difficulties. Not only is TPLSM shown to have much less photobleaching and better penetration depths than CLSM, but also it is capable of doing UV imaging without using special UV optical elements. Several different aspects of TPLSM are discussed in the dissertation. The progress of 3-D fluorescence microscopy, a historical retrospective of two-photon excitation, the physics of two-photon excitation and our instrument setupare is discussed in the first chapter. In chapter II, a qualitative and quantitative confirmation of two-photon excitation, optical transfer efficiency, point spread function and resolution, signal-to-noise ratio, and two-photon excitation spectrum are presented. Chapter III shows the comparison between TPLSM and CLSM. Although CLSM has slightly better resolution than TPLSM, TPLSM has much less photobleaching and toxicity, greater penetration depth, less signal cross talk, and better signal collecting efficiency. Image deconvolution techniques with CLSM and TPLSM are discussed in chapter IV. Using this image processing methods and acquired PSF, we improved the resolution of CLSM and TPLSM dramatically. These deblurring techniques were applied to study the positions of proteins in the pre- and postsynaptic compartments of rat hippocampal culture cells. The improved resolution enabled us to distinguish the positions of Synapsin I, CaM Kinase U, and PSD-95, which could only be done with electron microscopy before. In chapter V, image degradation due to brain tissue scattering is discussed. Emission signals with long wavelengths were shown to have better resolution and image contrast because of less tissue scattering. This result shows the necessity of designing fluorophores with low emission wavelengths for deep tissue imaging. TPLSM already provides many insightful images to the study of the dynamics and structures of biological systems. With the advance of technology, applications of TPLSM will be further developed to help understand biological or physical mechanism