Biomedical Engineering Reference
In-Depth Information
antibody was subsequently used to render labeled regions fluorescent. After de-
hydration and clearing, the specimens were mounted in double-sided custom
slides.
The brains were imaged with a confocal laser scanning microscope (Leica
TCS 4D). The chromophor was excited with an ArKr laser, and the fluorescence
was detected using a longpass filter. The intensity of the fluorescence was quan-
tized with a resolution of 8 bits. Due to the size of the dissected and embedded
brain (about 2
.
5
×
1
.
6 mm laterally and about 0
.
8 mm axially), it cannot be im-
aged in a single scan. Therefore we used multiple image-stack acquisition (3D-
MISA) [88]. The entire brain was scanned in 2
×
3 partially overlapping single
scans, each using 512
×
512 pixels laterally and between 80 and 120 sections axi-
ally. The stacks were combined into a single 3D image using custom software or
a script running in Amira (see next paragraph). Because of the refractive index
mismatch between the media in the optical path, images exhibit a shortening of
distances in axial direction that was accounted for by a linear scaling factor of
1.6 [7].
Post-acquisition image processing was done with the Amira 3D scientific
visualization and data analysis package (ZIB, Berlin, Germany; Indeed - Visual
Concepts GmbH, Berlin, Germany; TGS Inc., San Diego, CA). Image stacks were
resampled laterally to half of the original dimensions in order to increase dis-
play speeds and allow interactive handling of the data. The final image volume
contained 84-114 slices (sections) with thickness 8
m. Each slice had 610-749
pixels in
x
direction and 379-496 pixels in
y
direction with pixel size 3
.
8
m. In
most cases no further image processing was necessary. In a few cases unsharp
masking filters were applied in order to enhance contours.
Subsequently, for each brain an atlas of the neuropil areas of interest was gen-
erated by tracing them manually on each slice. We distinguished 22 major com-
partments, 20 of which are bilaterally symmetric on either brain hemisphere [39].
The paired structures we labeled were medulla, lobula, antennal lobe, ventral
mushroom body consisting of peduncle,
α
- and
β
-lobe, and medial and lateral
lip, collar and basal ring neuropil. The unpaired structures we identified were
the central body with its upper and lower division and the protocerebral lobes
including the subesophageal ganglion. Examples of confocal microscopy and
label images are shown in Fig. 11.1. Three-dimensional surface renderings of
the segmented bee brain are shown in Fig. 11.2. The labeled structures and the
abbreviations used for them in this chapter are listed in Table 11.1.
