An Integrated Micro- and Macroarchitectural Analysis of the Drosophila Brain by Computer-Assisted Serial Section Electron Microscopy
(A) The object hierarchy window displays segmentation data types (left column, green), all segmented objects captured with these types (center column, magenta), and all layers along the z-axis (right column, blue). (B) ImageJ tool box. (C) The 2D canvas presents the TEM (or any other) raw data stack. The user can scroll and navigate through all sections displayed in this window. The yellow grid at bottom left of panel shows all of the image tiles that constitute the montage of the section shown. When zoomed in, a red frame indicates the area shown on canvas; by dragging the red frame, the user can navigate to the desired parts of the montage. Segmentation and annotation of identified structures (e.g., compartments and lineage tracts) is performed in the 2D canvas window. Shown are outlines of four identified axon fascicle (APT, antenno-protocerebral tract, blue; deCP, descending central protocerebral fascicle, yellow; MEF, medial equatorial fascicle, red; p, peduncle of mushroom body, light blue). Points of intersections of fascicles, such as the one between deCP and MEF (blue circle), serve as fiduciary marks with which different stacks are registered. (D) The interactive 3D viewer windows show selected objects segmented from the TEM stack. Shown as examples are the same elements depicted as sections in panel C [mushroom body (CX, calyx; ml, medial lobe), MEF fascicle, deCP fascicle]. (E–J) Registration of confocal stack to TEM stack. (E) 3D digital model of larval brain hemisphere derived from confocal stack. Shown are brain surface (light gray), mushroom body (light blue), and axon fascicles (dark gray). Set of fascicles highlighted in TEM stack (see panels C, D) are shown in corresponding colors. (F) 3D digital model of mushroom body, showing location of fiduciary marks (yellow) identified in both TEM stack and confocal stack. (G) Representative section of TEM stack. Shaded in purple are profiles of two axon fascicles (p, peduncle; MEF, medial equatorial fascicle) as they appear in the TEM section. Superimposed in red and blue are profiles of corresponding fascicles from confocal stack that was registered with TEM stack (using fiduciary marks shown in F) and digitally re-sliced along the plane of sectioning of the TEM stack. Note high degree of overlap between TEM profiles and “imported” confocal profiles. (H) Representative section of confocal stack of one brain hemisphere. Shown in green are GFP-labeled PDF neurons. The cell bodies of these neurons are located in the dorso-lateral cortex; axons form bundle that extends along the dorso-lateral cortex-neuropile boundary (white dotted line). Large arrowhead points at one cell body, small arrowhead at axon. (I) Section of registered, re-sliced confocal stack projected upon corresponding EM section. Cell body and axon are indicated by large and small arrowhead, respectively; boundary between dorso-lateral cortex and neuropile is shown as white dotted line. (J) Magnified view of area boxed in (I). Arrows point at profiles of neurites containing (neuro-secretory) dense-core vesicles. Based on their proximity to the position of PDF neurons, it is probable that these profiles correspond to branches of PDF neurites. Scale bars: 5 µm (C, G, I); 10 µm (H); 1 µm (J).