Fluorescence Imaging Of Golgi-Cox Stained Mouse Brain Neuron

In the study of brain wiring, especially abnormal connection of neuron network in brain diseases, Golgi method, as a classical labeling tool, is commonly used to research the primate and human brain. During specimen preparation after the Golgi method, the millimeter-level thick tissue usually has been cut into micron-level brain slice. However, due to the cutting depth of vibratome sectioning or crystat-sectioning, the thickness of brain slices are difficult to achieve less than ten microns. In wide-field or confocal microscopy, the thick section will lead to hiatus in3D reconstruction of whole brain data. The knife-edge line scanning imaging can image the sample on one micron brain slice, but it is slow in data acquisition. Therefore, developing the fast3D sectioning imaging method for Golgi-stained whole brain is hot nowadays.Golgi method can label neurons widely, but its mechanism is still unexplained accurately. The analysis of precipitates on Golgi-stained neuron is the material basis of understanding its staining mechanism. However, classical optical technology, e.g. wide-field transmitting or confocal reflection microscopy, can not analysis the components of stained neurons. Other chemical tests can not accurately locate on the stained neuron or achieve the purification of the precipitates on stained neurons. Therefore, employing new imaging method and multiple characterization tools for the component study of precipitates is useful for the Golgi-Cox-stained mechanism.The purpose of this thesis is to develop fluorescence imaging methods for Golgi-Cox-stained mouse brain neuron, and to combine multiple methods to analyze the precipitates on stained neuron, which will expand the application of Golgi-stained neuron and promote the understanding of Golgi mechanism.The single photon fluorescence imaging of Golgi-Cox-stained mouse brain neurons has been studied. Firstly, the fluorescence spectrum of background from the whole mouse brain prepared by modified Golgi-Cox method has been analyzed. Next, the principle of fluorescence imaging was suggested and tested on both wide-field and confocal fluorescent microscopy. By comparing the conventional Golgi-staining imaging and single photon fluorescence imaging approach, the latter can effectively prevent background. For this, the soma of stained and unstained neurons from the same coronal section can be distinct by confocal fluorescence imaging.The wide-field fluorescence imaging has been developed into wide-field fluorescence sectioning imaging for Golgi-Cox stained mouse neurons. The capability of wide-field fluorescence sectioning imaging has been discussed by imaging processing based on wide-field fluorescence microscopy and the strategy of imaging first and cutting-off later. After data acquisition and analysis, the3D wide-field fluorescence sectioning images have been achieved for Golgi-Cox-stained mouse brain neurons.The two-photon fluorescence imaging of Golgi-Cox stained mouse neurons has been carried out. Combining micro-infrared spectrum, the fluorescence emitting principle of stained neuron has been explored. The precipitates on stained neuron have been analyzed by SEM, ESEM, EADX, and XPS for morphology, element, element state and functional group. The correlation of Hg-binding protein on precipitates and Golgi-Cox-stained mechanism has been discussed. The precipitates on Golgi-Cox-stained neuron have been discussed before and after alkalization process.