| Fast and high-resolution imaging of brain-wide neuroanatomical architecture of Drosophila melanogaster is essential to understand brain function and dysfunction. However, limited by low axial resolution, traditional optical imaging methods used to map the Drosophila brain at sub-micron resolution in two dimensional planes. Recently, an emerging optical imaging technology based on "sectioning-imaging" method, represented by the micro-optical sectioning tomography (MOST) system, takes both advantages of high-spatial resolution and fast imaging speed and brings new developmental opportunities to the field of neuroanatomy. To visualize the Drosophila whole-brain at a three-dimensional high-resolution, the present study focuses on developing a sample preparation method, and further a cell type-specific labelling method, both of which are suitable for sectioning-imaging of the fly whole-brain.According to technical requirements of the sectioning-imaging approach, a preparation method for the Drosophila whole-brain sample was studied. The processing procedures of the sample were set as fixation, staining, dehydration and embedding in order, which were different from traditional histological methods, and staining and embedding were the two key steps. For the staining method, to reveal brain-wide neurons and neuronal processes in Drosophila, the Golgi method was investigated and optimized, and uniform and moderate staining effect was attained through the fly whole-brain. For the embedding method, to make the sample suitable for continuously sectioning in micron thickness, one epoxy resin named Spurr was taken as the embedding medium, and the experimental conditions of embedding and dehydration were tested and optimized. Based on the above experimental results, the present study set up a set of methodology based on Golgi-staining method for preparing fly whole-brain sample that is suitable for the sectioning-imaging approach.In order to remedy the randomness of labeling of the Golgi-staining method, a cell type-specific labelling sample preparation method was developed. The immunoenzyme method based on horseradish peroxidase/diaminobenzidine (HRP/DAB) provides high-contrast images to resolve details underlying specific neuronal architecture. However, the poor staining penetration of the HRP/DAB staining restricts its application to the detection of neurons located in the surface layer. The present study modified the HRP/DAB method to obtain uniform brain-wide staining. Combining the sample processing protocol and the embedding method established above, the fly whole-brain sample labelling specific neurons was prepared.Finally, the present study demonstrated preliminary applied research on sectioning-imaging of the Drosophila whole-brain. By means of the MOST system, brain-wide neuroanatomical architecture of Drosophila was fast and high-resolution imaged and three-dimensional data sets were acquired at a submicron voxel resolution. In the Golgi-stained fly whole-brain data set, brain-wide neural cells and neurites of Drosophila were visualized in three-dimension, and some underlying neural cell types and projections were found, such as the long-distance neuronal projection from antennal lobe to lobula complex, which may afford anatomical evidences for crossmodal interaction among different functional regions. In the IE-stained fly whole-brain data set, the morphological structures of the Drosophila serotonergic neurons were revealed at cellular resolution in brain-wide scale, and some unique neuronal structures were provided rather than the traditional imaging method and could, thus, be a valuable comparison data for more specific, targeted studies.In summary, this study demonstrated fast and high-spatial-resolved imaging neuroanatomical architecture in the Drosophila whole-brain, provided a novel method to reveal brain-wide neuronal networks of Drosophila, and could play a promoting role in research on brain structure-function relationships. |
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