Japanese Researchers from the RIKEN Center for Developmental Biology Create New Optical Clearing Agent; Neurolucida Used For 3D Imaging in Study
A new optical clearing agent developed by scientists in Japan clears brain tissue samples with greater transparency and less time than other clearing agents, according to a paper published in Nature Neuroscience.
“Combined with two-photon microscopy, SeeDB allowed us to image fixed mouse brains at the millimeter-scale level,” say the authors, who after clearing the brain tissue with SeeDB, captured images with a multiphoton Olympus microscope, and visualized 3D reconstructions with Neurolucida.
A solution of fructose, water, and alpha-thioglycerol, SeeDB cleared gray and white matter brain tissue samples in three days without affecting the volume or morphology of the tissue. Dendritic spines of pyramidal neurons in the cerebral cortex was one aspect of fine morphological architecture that the authors note remained intact after SeeDB treatment.
Compatible with fluorescent proteins and various neuronal tracers including lipophilic dyes such as DiI, the clearing agent proved to be an effective tool for quantitative reconstructions of microcircuits in the brain. To illustrate this point, the authors examined a detailed wiring diagram in the mouse olfactory bulb. They labeled neurons with fluorescent dextran tracers, cleared the tissue with SeeDB, and using confocal microscopy, imaged the wiring diagram of the dendrites of a type of neuron known as mitral cells. To reconstruct these dendrites, the scientists analyzed confocal images and manually traced the dendrites in three-dimensional space using Neurolucida.
“SeeDB is particularly advantageous for quantitative analyses of fine neuronal morphology and microcircuits because minimal deformation artifacts occur during the clearing process,” the authors say. “Because our protocol is quick, easy, inexpensive, safe and requires no special equipment, SeeDB could prove useful for a broad range of researchers working in neuroscience and developmental biology.”
Ke, M., Fujimoto, S., Imai, T., (2013). SeeDB: a simple and morphology-preserving optical clearing agent for neuronal circuit reconstruction. Nature Neuroscience, doi:10.1038/nn.3447