Study On A Plastic-Embedding Method Of Large Samples For High-Resolution Fluorescence Imaging

The complete acquisition of fine structure and protein distribution of neurons throughout whole brain is important for understanding the mechanism of complex neural connections and neural signaling transduction in the brain.The developments of fluorescent labeling technologies provide powerful tools for the visualization of the structure and protein distribution of brain neurons,and also promote the progress of large volume imaging techniques.Based on high resolution imaging and precision continuous sections,Micro-Optical Sectioning Tomography technology can image the whole mouse brain with high spatial resolution.We have successfully obtained 3D high-resolution imaging datasets of the fluorescent protein-labeled whole mouse brain with submicron resolution,which arise wide attention both at home and abroad.One of the key to this progress is to embed fluorescent protein-labeled whole mouse brain before imaging,which facilitates precise slicing and high-resolution imaging.For a cubic centimeter-sized sample,plastic-embedding method currently used has improved the fluorescence preservation of sample.However,it is hard to prepare sample with stable quality and high efficiency,and the existing large-sample embedding method may cause fine structure loss.On the other hand,it is not clear whether the large-volume sample embedding method is compatible with entire block immunostaining technology.Therefore,this paper focused on sample preparation,and optimized a plastic embedding method based on the existing methods,which can better preserve fine structure in whole mouse.A standardized procedure of sample preparation was established,which can improve stability and repeatability of sample preparation.The compatibility of large volume immunofluorescent labeling technique and plastic embedding was examined.A plastic embedding method which is suitable for large volume immunofluorescent labeled sample was established.The main research contents are as follows:(1)Study on the embedding method of maintaining fine structure of fluorescent protein labeled mouse brain.We tested and optimized the plastic embedding technology of fluorescent protein labeled large samples,and found out that prolonging the fixation time to 48 hours will help minimize the distortion of neurite structure.it is favorable to maintain the fluorescence signal of neurite structure if the dehydration is processed in 4 ? environment.Lowicryl HM20 resin was chosen,and the polymerization process was optimized.This method could better retain the edge of mouse cerebral cortex fine structure such as apical dendrites,as well as the neuron membrane structure and subcellular structure.On the basis of this,a standardized plastic embedding process of whole brain is established.(2)Study on the preparation method of immunofluorescent labeled mouse cerebral for high resolution imaging.The project conducted a quantitative study of the fluorescence maintaining of immunofluorescent probes in three kinds of resins,and found that all probes fluorescence were well maintained in GMA,Lowicryl HM20 resins,while LR White had a relatively low maintenance rate than other resin.We also assessed the serial sectioning properties of resins and found that the Lowicryl HM20 resin demonstrate better slice properties.Then,the absorption spectra of different fluorescent probes before and after Lowicryl HM20 resin embedding was examined as well,which proved that the minor change of fluorescence absorption spectrum was not the main reason for the change of fluorescence intensity before and after resin embedding.Further study of compatibility between i DISCO staining technique and Lowicryl HM20 resin embedding was also conducted,and discovered that this embedding method can be well compatible with a variety of fluorescent antibodies and fluorescent tracers labeled brain tissues.Finally,we embedded the immunofluorescent labeled whole mouse brain and monkey cerebral cortex for high-resolution imaging,and acquired 3D high-resolution images.The established plastic-embedding procedure was reliably used in many domestic and foreign research groups,and related research works was promoted.This embedding method can not only be applied to neuroscience,but also to other fields of life science with vital significance.