BrainAligner is an automatic computer program that registers a (or any) pair of 3D image stacks using landmark matching. It has been primarily used in Janelia Farm’s FlyLight project to align many thousands of laser scanning microscope images, each of which has hundreds of millions pixels and multiple fluorescent colors that highlight various subsets of neurons in a brain as well as the overall brain morphology. 



  1. Mac

  2. Linux (Redhat/Fedora)

  3. README: a VERY SIMPLE instruction on how to run the program

Source Code

  1. svn co

Test Data

We have put here some test images. If you need additional information, please send me an email (pengh<at>

  1. Image 1 (6.8M bytes, TIF format)

  2. Image 1 sample landmarks (plain text file, CSV format)

  3. Image 2 (6.8M bytes, TIF format)

One target brain image used in our large-scale atlas project (and the Nature Methods 2011 paper).

  1. Target image (M295) used in real experiments (43M bytes, zipped TIF)

  2. A landmark file used in the paper (note, for different applications, you may need to use different landmark files)

Examples of subject images in our large-scale atlas project. For these test data, only the NC82 reference channel is kept (and the neuronal patterns have been removed). Note, to align these example images to the above target image, you need to run the global alignment (with ‘BrainAligner -w 0’) and then local alignment (with ‘BrainAligner -w 10’), and optionally need my optic lobe segmentation program (Lam, et al, 2010. See below).

  1. 20x FlyLight Image 1 (thanks to Chris Zugates and his sample-prep team!)

  2. 20x FlyLight Image 2


Major publication:

  1. Peng, H., et al, (2011) “BrainAligner: 3D registration atlases of Drosophila brains,” Nature Methods,  Vol. 8, No. 6, pp.493-498, DOI: 10.1038/nmeth.1602, 2011. [PDF]

Related publications:

  1. Peng, H. et al, (2010), "V3D enables real-time 3D visualization and quantitative analysis of large-scale biological image data sets," Nature Biotechnology, Vol.28, No.4, pp.348-353, DOI:10.1038/nbt.1612, 2010. [ PDF | Software ]

  1. Qu, L. and Peng, H. (2010) "A principal skeleton algorithm for standardizing confocal images of fruit fly nervous systems," Bioinformatics, Vol.26, No.8, pp.1091-1097, 2010. [ PDF | Supplementary Videos, Figures, Programs, and Data | Software checkout: "svn co"]

  1. Lam, S. ..., Peng, H., (2010) "Segmentation of center brains and optic lobes in 3D confocal images of adult fruit fly brains," Methods, Vol.50, No.2, pp.63-69, 2010. [PDF | Software checkout: "svn co"] 

Other Links

  1. Vaa3D and Vaa3D-AtlasViewer:  viewing the images and registration results

  2. Developer’s homepage: Hanchuan Peng.


You can right-click the following links to download the program and test data ...

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