CLARITY (Clear, Lipid-exchanged, Anatomically Rigid, Imaging/immunostaining compatible, Tissue hYdrogel) is a treatment for the transformation of intact tissue, here brain tissue, into a nanoporous hydrogel-hybridized form that is optically transparent and macromolecule-permeable.
Whit this method is possible to obtein a fully assembled tissue for studing of long-range projections, local circuit wiring, cellular relationships, subcellular structures, protein complexes, nucleic acids and neurotransmitters using in situ hybridization and immunohistochemistry with multiple rounds of staining and de-staining.
The protocol provide to infuse hydrogel monomers (here, acrylamide and bisacrylamide), formaldehyde and thermally triggered initiators into tissue at 4° C. In this step, formaldehyde not only crosslinks the tissue, but also covalently links the hydrogel monomers to biomolecules including proteins, nucleic acids and small molecules.
Next, polymerization of the biomolecule-conjugated monomers into a hydrogel mesh is thermally initiated by incubating infused tissue at 37° C for 3 h, at which point the tissue and hydrogel became a hybrid construct. Importantly, lipids and biomolecules lacking functional groups for conjugation remain unbound and therefore can be removed from the hybrid.
To extract lipids efficiently requires an active transport organ-electrophoresis approach here electrophoretic tissue clearing (ETC). ETC is based on the use of a custom-built organ-electrophoresis chamber in which circulates a solution of sodium borate buffer (200 mM, pH 8.5) containing 4% (wt/vol) SDS with a voltage of 10-60 V across the brain at 37–50° C for 2 days.
ANALYTICAL TRICKS AND TIPS
Since the imaging depth in clarified tissue is limited by the working distance of the objective it could be useful to acquire half of the brain and then turn it and acquire the second half. in this way is possibile to reach the maximum of the imaging of the tissue.
Clarified tissue can be analyzed with several conventional microscopy method such as light mircoscopy, confocal microscopy, two-photon microscopy and single photon mircoscopy. The latter is to be preferred because many dyes and fluorescent proteins are better suited to single-photon rather than two photon illumination; while the confocal illumination expose the entire depth of tissue to excitation light and therefore induces substantial photobleaching of fluorescent molecules.
A portion of tissue can be extracted for electron microscopy. Clarified tissue preserves some
ultrastructural features such as postsynaptic densities, although because of the absence of lipid, conventional electron microscopy staining does not currently provide enough contrast to identify all relevant ultrastructures and boundaries.
THE BIOLOGICAL CONTEXT
Clarified tissues can be analized with different microscopy method with the same achievement and limitation and the improvement of show the whole tissue. It is possibile to investigate cellular relationships, subcellular structures, protein complexes, nucleic acids and neurotransmitters. Furthermore allows molecular phenotyping of intact-tissue volumes and tracing of structure such as neuron projection.
Using CLARITY in combination with immunofluorescence is possibile to perform serveral phenotypic analysis such as viewing a single neuron population and their projection. With the antybody against Tyrosine all catecholaminergic neurons are labeled allowing to observe the arrangement in the whole brain. (Structural and molecular interrogation of intact biological systems)
In addition, the combination of CLARITY with genetic methods for identification of synapses or for labeling specific circuits is also of interest. The GRASP method is an excellent example. In this method, two nonfluorescent split-GFP fragments can be virally expressed in the synaptic
membrane of two separate neuronal populations; these two fragments reconstitute fluorescent GFP only across a synaptic cleft so that the location of synapses between the two populations can be visualized. (GFP Reconstitution Across Synaptic Partners (GRASP) Defines Cell Contacts and Synapses in Living Nervous Systems) These versatile targeting approaches, in combination with CLARITY, could provide a high-throughput approach for globally mapping synaptically connected and synaptically activated populations across the brain that could complement the use of electron microscopy in some settings. (Clarity for mapping the nervous system)
Clarified tissue allows to avoid the mechanical sectioning methods that can cause deformation of tissue and uncertainty in registration across sections. Therefore it can be a crucial method for the analysis of neurological pathologies such as autism-spectrum disorder or down sindrome.
- Allows to analyze the whole brain
- Preserve most of the biological molecules
- Possibility of multiple rounds of staining and de-staining
- Compatible with common analysis method
- Cheap and easy to perform
- Loss of biomolecules lacking functional groups
- Compatibility incomplete with electron microscopy for lacking of lipid
- Lack of appropriate virtual reconstruction programs
With CLARITY 8% of total protein content is lost but the nature of the remaining should be investigated. Therefore is unknow how long clarified tissue may be maintained or stored for such analysis, assessment, imaging or remodeling in subsequent rounds of CLARITY processing.