Three-dimensional Reconstruction of the Drosophila Larval and Adult Brain
- Materials and Methods
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 (Switze
rland).
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 4ºC. 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 µm for this setup. According to the
sampling theorem we chose to sample at about 1 µm in all directions, thereby accepting an
undersampling in the lateral plane (as the lateral resolution is better than 2 µm). This
resulted in image stacks with a size of 535 µm x 535 µm x 195 µm, 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, http://amira.zib.de) 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).
Larval Brain Model
Adult Brain Model
Main
| The Flybrain
| About 3D Reconstruction
| Handling of 3D Models