A transparent 3-D view of amyloid beta plaques (green) and blood vessels (red) in a region of cerebral cortex from a 20-month-old AD model mouse (credit: RIKEN)
Researchers at the RIKEN Brain Science Institute in Japan have created a new technique for converting brain tissue into transparent tissue to reveal 3D brain anatomy at very high resolution.
The researchers say they have used the new technique, called ScaleS, to provide new insights into Alzheimer’s disease plaques and for large-scale connectomic mapping and 3D neural circuit reconstruction.
Three-dimensional view of stained hippocampus showing fluorescent-expressing neurons (green), connecting interneurons (red) and supporting glia (blue), created with the Stanford CLARITY technique (credit: Deisseroth lab)
Previous techniques, such as Stanford’s CLARITY, for creating transparent brain samples — a process called “optical clearing — are useful for microscopy, but the transparency process itself can damage the structures under study, the researchers note. The structures are also not firm enough for electron microscopy, which is used to provides images at a finer level.
These limitations are now overcome with ScaleS, according to the researchers. The internal structures also maintain their original shape and are firm enough to permit the micron-thick slicing necessary for more detailed analyses.
Published in Nature Neuroscience, the new technique combines sorbitol in the right proportion with urea. “We could create transparent brains with minimal tissue damage,” said lead scientist Atsushi Miyawaki. The technique can handle “both florescent and immunohistochemical labeling techniques, and is even effective in older animals,” he added.
Miyawaki believes that the quality and preservation of cellular structures viewed by electron microscopy with ScaleS is unparalleled.
New Alzheimer’s disease findings
A 2-D version of a 3-D image of amyloid beta plaques in entire brain hemispheres of an Alzheimer’s disease model mouse created with ScaleS. The inset (bottom right) shows a high-magnification volume rendering of a representative senile plaque (credit: RIKEN)
The researchers were able to use ScaleS to visualize the mysterious “diffuse” plaques seen in the postmortem brains of Alzheimer’s disease patients that are typically undetectable using 2D imaging. They found that contrary to current assumptions, the diffuse plaques proved to be not isolated; they showed extensive associations with microglia — mobile cells that surround and protect neurons.
The researchers also examined 3-D images of active microglial cells and amyloid beta plaques. While some scientists suggest that active microglial cells are located near plaques, a detailed 3D reconstruction and analysis using ScaleS clearing showed that association with active microglial cells occurs early in plaque development, but not in later stages of the disease, after the plaques have accumulated.
“Clearing tissue with ScaleS followed by 3D microscopy has clear advantages over 2D stereology or immunohistochemistry,” states Miyawaki. “Our technique will [also be useful] for examining normal neural circuits and pinpointing structural changes that characterize other brain diseases.”
Abstract of ScaleS: an optical clearing palette for biological imaging
Optical clearing methods facilitate deep biological imaging by mitigating light scattering in situ. Multi-scale high-resolution imaging requires preservation of tissue integrity for accurate signal reconstruction. However, existing clearing reagents contain chemical components that could compromise tissue structure, preventing reproducible anatomical and fluorescence signal stability. We developed ScaleS, a sorbitol-based optical clearing method that provides stable tissue preservation for immunochemical labeling and three-dimensional (3D) signal rendering. ScaleS permitted optical reconstructions of aged and diseased brain in Alzheimer’s disease models, including mapping of 3D networks of amyloid plaques, neurons and microglia, and multi-scale tracking of single plaques by successive fluorescence and electron microscopy. Human clinical samples from Alzheimer’s disease patients analyzed via reversible optical re-sectioning illuminated plaque pathogenesis in the z axis. Comparative benchmarking of contemporary clearing agents showed superior signal and structure preservation by ScaleS. These findings suggest that ScaleS is a simple and reproducible method for accurate visualization of biological tissue.