Confocal and Multiphoton Imaging Core will host a demonstration of the Scale lens from Carl Zeiss. This LD Plan-Apochromat 20x/1.0 lens with working distance of 5.6 mm allows to carry out imaging of a whole brain cleared with the Scale technique, published last year in Nature Neuroscience by the group of Dr. Artsushi Miyawaki, Brain Research Institute, RIKEN, Japan. The lens is installed on the Zeiss 7MP Multiphoton Microscope in Rm. 8122. In Scale-treated mouse brain, neurons labeled with genetically encoded fluorescent proteins were visualized at an unprecedented depth in millimeter-scale networks and at subcellular resolution. The improved depth and scale of imaging permitted comprehensive three-dimensional reconstructions of cortical, callosal and hippocampal projections whose extent was limited only by the working distance of the objective lenses.
On the image below: a. A three-dimensional reconstruction of YFP-expressing neurons in 16 (8 × 2) quadratic prisms located in the cerebral cortex and hippocampus is shown in b. A high-magnification xy image at a depth of 0.9 mm (a yellow box in b) is shown in c. (Image taken from Hama et al., Nat. Neurosci. 2011)
Scattering is the major problem limiting light penetration into live tissues. Optical clearing allows to achieve refractive uniformity, leading to the reduction in scattering. Several clearing techniques have been developed earlier, of which benzyl benzoate/benzyl acolohol (BBA) allowed to achieve high level clearing, however, at the expense of significant quenching of fluorescent labels. Scale technique, based on urea, is claimed not to affect labels' quantum yield. Although an increase in brain size is observed at Scale application (BBA causes shrinking), relative positions of internal structures (hippocampus, amygdala and white matter) maintained their overall shape and proportions.
Clearing protocols and brain preparation
We have prepared both Scale/A2 and Scale/B4 clearing agents. They are available for everybody interested in trying out the technique.
Special thanks to the lab of Dr. William Snider for providing the reagents and to Jason Newbern in particular for his help.
Please contact Vladimir Ghukasyan to obtain the solutions.
When and Where
How to apply
If you would like to try the clearing/imaging, please contact Vladimir Ghukasyan:
Rm. 7109F NRB
Tel.: 919 966 5807
Note: 2 weeks are required to clear the brain. Please plan correspondingly