| The neuronal functional connections mapping of the brain has become a hot spot as well as a difficulty in modern neuroscience research. It is essential to get the neuronal functional connections in loop level and whole brain level, whether to explore the mysteries of the brain, or promote the diagnosis and treatment of brain diseases. However, the research tool to realize high precision imaging of big fluorescent labeling samples is still lacking, fast and high-precision fluorescence imaging method and system for large samples is urgent needed.In this thesis, we developed a novel method to realize high precision imaging of big fluorescent labeling samples. It combines two photon imaging and plastic embedding technology, because the sample was embedded in a plastic embedding manner, thus a high sampling accuracy of submicron could be obtained. Moreover, the focal plane was set to below the surface of the sample for the features of two photon imaging, together with the method of mechanic sectioning after imaging, the cutting requirements was significantly decreased and the integrity of data was maintained.Based on this novel method, we design and set up a two-photon fluorescence micro-optical sectioning tomography (2p-fMOST) system. The transmission efficiency of the system reaches16.5%with a maximum power of470mW after the objective lens, which can meet most needs of two-photon imaging studies. The resolution of the system is0.45μm in lateral and1.68μm in axial, which can image the spines of the neuron fibers clearly. The imaging area achieves50mm×30mm×20mm, which is big enough for whole mouse brain imaging. Moreover, in order to reduce adjusting complexity and improve image stability, we engineered the2p-fMOST system by modular design.Based on this2p-fMOST method and system, we acquired a three-dimensional data set of a Thyl-enhanced green fluorescent protein (Thyl-EGFP, M line) transgenic mouse brain with high precision. After215h, we generated this entire brain database with a voxel size of0.5×0.5×2μm3by6330optical sections. In this data set, spines and axons could be easily resolved. Besides, we traced several neural long-distance projections distributed in different brain regions based on this data set because of its high spatial resolution, high voxel resolution and high integrity. And the longest of these axons reached to12.5mm. In addition, this data set was also used in the application of automated localization of neurons for brain circuits by NeuroGPS method for its high signal noise ratio.The test shows that2p-fMOST system is a novel tool that can acquire neural functional connection data set in the whole brain with high precision, high speed and high integrity. The application shows that the2p-fMOST system has a great potential in neuron science. |
PDF Full Text Request