Friday, September 11, 2015

Using BrainAligner to warp z-stacks of Drosophila brains

In my recent paper on central complex processing during flight, I used BrainAligner to register z-stacks of Drosophila brains to one another. Getting it to work took a little fiddling, which I'll record below.

To install, follow directions here.

Next, right-click brainaligner_linux_redhat_fedora_64bit and choose properties. Under the Permissions tab, click the "Allow executing file as program" checkbox.

In terminal, type
gedit ~/.bashrc

and add
export PATH="/home/$USER/<rest of path to directory>:$PATH"

at the end of the file to add <rest of path to directory> to your path.

It gave me the following error
"error while loading shared libraries: cannot open shared object file: No such file or directory"
Based on this stackoverflow answer, I ran
sudo ln -s /usr/lib/x86_64-linux-gnu/ /usr/lib/x86_64-linux-gnu/
to get it to recognize
And it seems to work!

To work with multiple color channels, you have to go to image>type>RGB color to change a composite image to a 3 channel color image. BrainAligner will assume the first channel is the reference channel to use in the warping, unless you give it a different channel number as an argument.

After much testing, I found that the following command worked well:

 ./brainaligner_linux_redhat_fedora_64bit -t ./20150101/Z3_8b_c_b3.tif -s ./20150101/Z1_8b_c_b3.tif -o ./20150101/warpedZ1.tif -w 10 -B 341 -x 1 -z 1 -X 1 -Z 1

The file names of the image stacks remind me that after converting each z-stack to RGB color (image>type/RGB color in imageJ), I binned every three pixels. This resulted in stacks that were 341x341 pixels, and the x-y resolution was equal to the z resolution. BrainAligner worked in about 2 hours.

Definitely check the warped stack afterwards, because BrainAligner will fail if the initial two stacks are not very similar to begin with. (I was aligning two stack from the same animal before and after photoactivation, which was a relatively easy alignment problem.)

Using ImageJ to count cell bodies in photoactivatable GFP experiment

In the PNAS paper, I counted cell bodies that contained photoactivated GFP after photoactivating the fan-shaped body. To do this, I followed this procedure: Acquire z-stacks before and after photoactivation, then align the first stack to the second stack using brainAligner (instructions here).

Next, download and install the Cell Counter plugin for imageJ.

Open both z-stacks. Make sure they have the same dimensions. (If not, scale them.)
Go to Plugins>Cell Counter
initialize Cell Counter on the after-activation stack.
Make sure the Counter Window (after activation z-stack) has focus, then click Analyze>Tools>Sync Windows

Now you can go through and click cell bodies that are greener in the after-activation stack than the before-activation stack.

Tuesday, September 8, 2015

Functional divisions for visual processing in the central brain of flying Drosophila

My new paper is now available in the PNAS Early Edition! I don't want to give too much away, but it has something to do with the structure pictured below:

NINDS also published a blog post about the work!