Three-dimensional Reconstruction of the Drosophila Larval and Adult Brain
- Materials and Methods

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Larval Brain

Immunohistochemistry: Larval and adult brains were prepared in Ringer solution and immediately fixed in 4% Para-Formaldehyde (15-45 minutes). The brains were subsequently put into 1% NGS blocking solution for 30 minutes and incubated with primary antibody for 6-12 hours at 4 C Dilutions for the antibody synorf1: 1:100. We used a secondary antibody with Cy3 labeling (Jackson Immuno Research). Washing between all steps used phosphate buffered saline (PBS) with 0.4% Triton X-100 (PBT). Preparations were embedded in Vectashield (Vector Laboratories).

Confocal microscopy and 3D Reconstruction: A Leica TCS4D confocal microscopes equipped with an ArKr-laser was used for data acquisition. Series of complete optic lobes were scanned with a 40x objective (aperture 1.0) and comprised 182 images of 512x512 pixel resolution at 8 bit color depth (47MB for each channel). The data were three-dimensionally reconstructed on a SGI Octane MXI graphics workstation using the 3D visualization software Amira (Konrad Zuse Zentrum, Berlin). Brain structures were demarcated by hand in confocal image stacks to define labels for the different structures, thus producing polygon groups of the surfaces of the structures. The polygon data were exported to the Virtual Reality Modeling Language (VRML 2.0 utf8). The resulting surface models were further processed with Cosmoworlds (Cosmosoftware) and optimized for smooth handling on current desktop systems. Volume rendering for the movie was performed with the software Imaris from Bitplane (Switzerland).

Adult Brain

Immunohistochemistry: Flies were anesthetized and brains were dissected in Drosophila ringer by stripping off the head capsule including the eyes and the laminae. Intact brains were fixed overnight in 2% para-Formaldehyde at 4C. The neuropil was stained using a mouse monoclonal primary antibody (nc82) and a CY3.18 conjugated secondary antibody (Jackson Immuno Research).

Confocal microscopy: Optical sections were acquired using a Leica TCS-NT confocal microscope equipped with Leica objective lens with a numerical aperture of 0.7. We measured an axial resolution of about 2 m for this setup. According to the sampling theorem we chose to sample at about 1 m in all directions, thereby accepting an undersampling in the lateral plane (as the lateral resolution is better than 2 m). This resulted in image stacks with a size of 535 m x 535 m x 195 m, or 512 x 512 x 168 voxels respectively (about 42 MB).

Data analysis: All data analysis and visualization were done using the Amira Software (Konrad Zuse Zentrum Berlin, on graphics computers (Indigo 2 High Impact, Onyx2 Infinite Reality, Silicon Graphics). Brain structures were labeled manually with the help of interactive tools. Surfaces were generated and exported into the VRML format. The image data were resampled into a standard orientation (frontal plane parallel to the ellipsoid body plane). Volume rendering for the movie was again performed with the software Imaris from Bitplane (Switzerland).

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