After two weeks of treatment with Scale, this mouse brain is completely transparent.

After two weeks of treatment with Scale, this mouse brain is completely transparent. © H. Hama et al.

At RIKEN’s Brain Science Institute a new reagent that turns brain tissue transparent is allowing neuroscientists to visualize neural circuitry at previously unattainable depths.

For decades, the limits of available technology have thwarted scientists’ attempts to visualise the complex inner workings of the brain. Breakthroughs in optical microscopy technology and a rapidly growing arsenal of multi-coloured fluorescent proteins have given researchers potent new tools for brain mapping. There are still challenges, however: the dense tissue of the brain scatters light and limits the depth to which these imaging methods can penetrate.

Now, following the development of a reagent they call ‘Scale’, Atsushi Miyawaki and his colleagues at RIKEN can render brain tissues as clear as glass, in a reversible transformation that gives researchers an unobstructed view of fluorescently labelled cells within.

The idea for Scale came from a chance observation of membranes made of polyvinylidene fluoride. This plastic material is usually white, but becomes completely transparent when soaked in concentrated urea. By tinkering with the solution, Miyawaki and colleagues came up with a mixture that has a similar effect on biological tissues.

                                                            Going deeper

Fluorescently labelled neurons

Fluorescently labelled neurons within the brain’s cerebral cortex and hippocampus. © Atsushi Miyawaki

Scale can render a mouse brain completely transparent within two weeks, but at the same time cells within Scale-treated samples fully retain their fluorescent labels. The transparency induced by Scale is now allowing researchers to explore deeper within the brain than ever before.

“Although the imaging depth limit of fluorescence microscopy is usually around 0.7mm in the brain, we were able to image fluorescent neurons with Scale down to a depth of 2mm below the brain surface,” says Miyawaki. A specialised lens enabled them to go even deeper, imaging at a depth of 4mm.

The level of detail enabled the team to analyse the interaction between neural stem cells and blood vessels within a developing mouse brain. They could also visualise neurons in the bridge between the brain’s two hemispheres.

Importantly, the effects proved to be fully reversible, and samples that had recovered from Scale treatment proved indistinguishable from their untreated counterparts, affirming Scale’s minimal impact on tissue structure.

A clear view of the future

Miyawaki and his team are already planning to use Scale for further investigations in mice. Although existing work has focused on genetically expressed fluorescent markers, the approach should be compatible with other labelling methods.

Scale could, for example, be used to work with larger tissue samples from species like primates that are not suitable for genetic modification. Scale’s biggest limitation at present is that its use is restricted to dead tissue, but Miyawaki suggests even this may change, saying, “At some point in the future, there may be ‘live Scale’!” Watch this space.

For further information contact:

Dr Atsushi Miyawaki
RIKEN Brain Science Institute, Wako, Japan