Human Brain Mapping Abbreviations

Fig. 1: Three-dimensional rendering of a clarified mouse brain (Chung et al. 2013)

To date, mapping of the brain is possible only in limited ways. Due to light scattering by the tissue, conventional deep tissue imaging by multiphoton microscopy is limited to around 1 mm tissue depth. To investigate brain structure and cell networks located in the center, the sample has to be sectioned and investigated in small volumes. Single neuron projections need to be followed across several sliced samples. This is elaborate. New tools are constantly being developed: automated sectioning methods reduce tedious work and minimize tissue damage, and novel software facilitates analysis and reconstruction. However, reconstruction may not always be possible or sufficient for detailed analysis of neuronal circuits. New optical clearing methods using organic solvents reduce light scattering and image deeper into the tissue. But these methods leave the lipid bilayers intact, so that they still function as diffusion barrier. Penetration of light and macromolecules for whole mount staining methods is still limited.

CLARITY (an acronym for Clear Lipid-exchanged Acrylamide-hybridized Rigid Imaging / Immunostaining / in situ-hybridization-compatible Tissue hYdrogel) is as simple as it is brilliant. The intact tissue is transformed into a hydrogel-tissue hybrid. Similar to petrification or fossilization, the physical structure of the tissue remains intact and is prevented from disintegrating. Light-scattering lipids are removed, while proteins and nucleic acids are preserved, because they are covalently linked to the hydrogel mesh.

In a first step, the tissue is infused with acrylamide/bisacrylamide, formaldehyde and a thermo-sensitive starter at 4 °C (Figure 2). Formaldehyde crosslinks the tissue and covalently links the hydrogel monomers to proteins and nucleic acids. Lipids and biomolecules lacking the functional groups are not bound and thus, can be removed. Only 8 % of proteins are lost by this method (compared to 25–40 % with conventional fixing and solubilization). Polymerization is triggered by elevating the temperature to 37 °C. The tissue is now a hydrogel-hybrid. The mesh supports tissue structure and its nanopores allow macromolecules to enter.

Lipid bilayers are removed by electrophoretic tissue clearing (ETC). In a custom-made electrophoresis chamber, the sample is placed in an ionic detergent (4 % SDS, sodium dodecyl sulfate) and an electric field is applied. The highly charged ionic SDS-micelles remove lipids actively. ETC has two major advantages above standard organic solubilization:

  1. It does not quench fluorescence like many organic solvents.
  2. Removal of lipids is fast. Passive diffusion of detergents would take up to months to completely remove the fat.

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