Reconstructions from Brainbow tissue
To understand how much of the dendritic material is obscured in Brainbow labled cells of the central nervous system compared to other labeling techniques, we can investigate each individual reconstruction and obtain arbor metrics using Neurolucida and Neurolucida Explorer (MBF Bioscience). Here are examples of five cells. While cells 1, 3 and 4 appear to have a complete dendritic arbor, cell 2 and 5 are clearly underrepresented due to many of their finer branches being obscured in the Brainbow tissue. In fact, those Brainbow cells that exhibit green fluorescence are of highest quality, while other fluorophores exhibit weaker and unstable labling.
The Brainbow tissue was bought from Jackson Laboratories. For more information on the Brainbow system the Lichtman Lab at Harvard Univeristy has a collection of reports at the following link: http://lichtmanlab.fas.harvard.edu/papers
Brainbow tissue was processed by Hermina Nedelescu, cut at 80um thick slices and mouted in Prolong mounting media. The slides were imagined with an Olympus confocal by Hermina Nedelescu and Alanna Watt with a 0.5um step size and a minimum of 10% overlap during acquisition in order to montage the tiles. Tiles were stictched by Hermina Nedelescu using Neurolucida's Montage Module. All images were deconvolved uisng AutoQuant Deconvolusion Software by Media Cybernetics. For more information on these parameters, please contact me.