Dual-mode Whole-brain Optical Imaging For Simultaneously Obtaining Golgi-stained Neuron Morphology And Orientation

Morphological analysis is necessary to understand the structure and function of brain,which can reflect the neural structure of brain function.Golgi-stained method is a classical morphological staining method,it is widely used to study neuron morphology in the complex neural networks.Especially for primate brain tissue,where the lack of other efficient labelling technologies,Golgi staining is still an indispensable stained method for the study of primate neuron morphology.However,as the short of suitable cytoarchitecture staining and obtaining method,although Golgi-stained method can acquire the dendritic morphology of individual neurons,the lack of accurate co-located information limit the application in the precise analysis of neuronal structure.Therefore,it is a technical challenge to provide a fast and high throughput imaging method to simultaneously obtain whole-brain Golgi-stained neuron signal and co-localization information.According to the requirement,this paper investigated the counterstaining feasibility of Golgi-stained thick tissues by the red fluorescent nucleic acid dye.By the comparison of the cytoarchitecture between Golgi-stained brain tissue and fluorescent samples,the feasible counterstaining of Golgi staining brain by fluorescent nucleic acid dyes was confirmed.Further,by the study of the permeability of red fluoscent nucleic acid dyes in Golgi-stained samples,and the effect of counterstaining quality on the axial imaging depth,the high contrast,high efficiency and single-cell resolution anatomical localization information was counterstained and obtained.Based on the counterstaining method,this paper developed a dual-mode micro-optical sectioning tomography.With the use of triple-window filter,simultaneous detection of dual mode signals was realized skillfully,which improved the efficiency of fluorescent/refect whole-brain imaging.By the study of sectioning performance of the resin embedded Golgistained brain tissue,the cutting device was improved,and the relationship of rapid section and data integrity was revealed.Furthermore,to adapt to different imaging purposes,two kinds of whole-brain imaging method,complete acquisition and fast acquisition method,were obtained.Though further optimization of the system,the Golgi-stained neuronal morphology and co-located cytoarchitecture of macaque monkey brain tissue was simultaneously obtained,which verified the system's wide-range and single-cell resolution dual-mode signal acquiring ability.By taking Alzheimer's disease as an example,under the precise anatomic localization,Golgi-stained method was used to carry out a model study on the neuron morphological analysis in different brain regions of specific brain diseases.To study the Alzheimer's disease mouse model and its control samples,hippocampus region was carried out.With the co-located anatomical annotation,three sub-regions in the hippocampus were accurately carried out.The quantitative analysis of neuron topological and metrical features were obtained in those sub-regions,and the morphological difference between Golgi-stained diseased and normal neurons were contrast,and the degradation degree of those neurons in the hippocampus of Alzheimer's disease was investigated.