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BrainAligner: 3D registration atlases of Drosophila brains

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Analyzing Drosophila melanogaster neural expression patterns in thousands of three-dimensional image stacks of individual brains requires registering them into a canonical framework based on a fiducial reference of neuropil morphology. Given a target brain labeled with predefined landmarks, the BrainAligner program automatically finds the corresponding landmarks in a subject brain and maps it to the coordinate system of the target brain via a deformable warp. Using a neuropil marker (the antibody nc82) as a reference of the brain morphology and a target brain that is itself a statistical average of data for 295 brains, we achieved a registration accuracy of 2 μm on average, permitting assessment of stereotypy, potential connectivity and functional mapping of the adult fruit fly brain. We used BrainAligner to generate an image pattern atlas of 2,954 registered brains containing 470 different expression patterns that cover all the major compartments of the fly brain.

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Figure 1: BrainAligner registers images of neurons from different brains onto a common coordinate system.
Figure 2: Schematic illustration of the BrainAligner algorithm.
Figure 3: Stereotypy of neuronal morphology and reproducibility of GAL4 expression patterns.
Figure 4: Expression pattern overlap by computational and biological methods.
Figure 5: Comparison of computational alignment of separate brains with coexpression data in the same brain.
Figure 6: A 3D atlas of neurite tracts reconstructed from aligned GAL4 patterns.

Change history

  • 16 May 2011

    In the version of this article initially published online, there was a misspelled word in the title and an incorrect callout to Figure 1e in the text. The errors have been corrected for the PDF and HTML versions of this article.


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We thank G. Rubin, B. Pfeiffer and K. Hibbard (Janelia Farm Research Campus, Howard Hughes Medical Institute) for the pJFRC21-10XUAS-IVS-mCD8::RFP, lexAop-CD2-GFP stock and LexAP vector; B. Ganetzky for collaboration in generating the GAL4 collection; B. Lam for visually scoring the quality of aligned brains; Y. Zhuang for tracing and proofreading neurite tracts; Y. Yu and J. Yang for manual landmarking; C. Zugates and members of the FlyLight project team for discussion of aligner optimization; G. Wu for help in testing a registration method; D. Shen for discussion when we initially developed BrainAligner; and G. Rubin for commenting on this manuscript. This work was funded by Howard Hughes Medical Institute.

Author information

Authors and Affiliations



H.P. designed and implemented BrainAligner, and performed experiments and analyses. H.P. and J.H.S. designed the biological experiments. F.L. and E.W.M. helped design the algorithm. P.C. prepared the samples and acquired confocal images. L.Q. helped implement the random sample consensus algorithm and some comparison experiments. A.J. produced the brain compartment label field. A.M.S. and J.H.S. generated LexA lines. H.P., E.W.M. and J.H.S. wrote the manuscript.

Corresponding author

Correspondence to Hanchuan Peng.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–6 and Supplementary Note 1 (PDF 21447 kb)

Supplementary Video 1

Aligned and overlaid neuronal patterns of a64-GAL4 and a74-GAL4. Magenta, a64-GAL4; green, a74-GAL4. (MOV 2728 kb)

Supplementary Video 2

Six aligned and overlaid GAL4 patterns in Figure 1d (see Fig. 1d for color scheme). (MOV 17577 kb)

Supplementary Video 3

The 3D reconstructed neurite tracts from 20 aligned a278-GAL4 images, along with their mean tract model (red). (MOV 2834 kb)

Supplementary Video 4

The aligned patterns of a156-GAL4; UAS-mCD8-GFP and LexAP078; lexAop-mCD2-GFP. Magenta, a156-GAL4; green, LexAP078. (MOV 3703 kb)

Supplementary Video 5

The co-expressed patterns of a156-GAL4; UAS-mCD8-GFP and LexAP078; lexAop-mCD2-GFP. Magenta, a156-GAL4; green, LexAP078. (MOV 6071 kb)

Supplementary Video 6

Aligned and overlaid patterns of CG8916_1-3-X-GAL4 along with its two Flp-out segments. Magenta, the original pattern; yellow and green, the Flp-out patterns. (MOV 4868 kb)

Supplementary Video 7

Maximal intensity project view of six aligned GAL4 patterns in the central complex (see Fig. 1d for color scheme). (MOV 10832 kb)

Supplementary Video 8

Cross-sectional view of six aligned GAL4 patterns in the central complex (see Fig. 1d for color scheme). (MOV 23447 kb)

Supplementary Video 9

A database of 269 stereotyped neurite tracts throughout the Drosophila brain. The width of each tract equals the respective spatial deviation. The tracts are color-coded randomly for better visualization. (MOV 15848 kb)

Supplementary Software 1

Brain Aligner and V3D Atlas viewer. (ZIP 9834 kb)

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Peng, H., Chung, P., Long, F. et al. BrainAligner: 3D registration atlases of Drosophila brains. Nat Methods 8, 493–498 (2011).

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