CLARITY Technique

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CLARITY has shown to be a viable method for intact tissue imaging of long-range projections, local circuit wiring, cellular relationships, subcellular structures, protein complexes, nucleic acids, and neurotransmitters. Intact tissues processed with CLARITY have also shown compatibility with
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CLARITY has shown to be a viable method for intact tissue imaging of long-range projections, local circuit wiring, cellular relationships, subcellular structures, protein complexes, nucleic acids, and neurotransmitters. Intact tissues processed with CLARITY have also shown compatibility with ''in situ'' hybridization and antibody labelling techniques, even supporting multiple rounds of staining and de-staining.
   
 
==Advantages and Disadvantages==
 
==Advantages and Disadvantages==

Revision as of 00:53, 20 April 2014

CLARITY is a technique developed in the Deisseroth lab at Stanford University.[1][2] The method is used to transform an intact tissue into an optically transparent and permeable hydrogel-hybridized form that can undergo immunostaining and high resolution 3-D imaging without damage to the sample. By clearing the tissue while preserving fine structural details, CLARITY provides a technique for obtaining high-resolution information from complex systems while maintaining the global perspective necessary to understand system function.

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CLARITY processing on a whole mouse brain

Contents

Methodology

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A schematic of the CLARITY process on a molecular level.


Biomolecules within the tissue are covalently linked to acrylamide monomers via reaction with formaldehyde. The acrylamide is thermally polymerized to form a hydrogel network crosslinked by the attached biomolecules and a small chemical crosslinker. An electric field is applied to actively transport SDS micelles through the tissue where they collect and remove the unattached lipids, leaving only the crosslinked biomolecules inside a swollen hydrogel matrix.


CLARITY has shown to be a viable method for intact tissue imaging of long-range projections, local circuit wiring, cellular relationships, subcellular structures, protein complexes, nucleic acids, and neurotransmitters. Intact tissues processed with CLARITY have also shown compatibility with in situ hybridization and antibody labelling techniques, even supporting multiple rounds of staining and de-staining.

Advantages and Disadvantages

Advantages

  • No damage or thin sectioning required to visualize whole intact tissue samples
  • Allows marking and visualization of long-range projections and subcellular structures
  • Allows multiple rounds of molecular phenotyping

Disadvantages

  • Multiple-step process that takes place over several days/weeks
  • Immunostaining is time-consuming for thicker tissue samples
  • High start-up and consumable material costs

Tissue Samples

CLARITY can be performed on a variety of tissue types. Challenges may arise from tissues that are more fibrous (limited porosity). Tissues that contain light-scattering elements other than lipids, such as pigments, may require additional clearing mechanisms to remove those elements.

Tissues that have been successfully cleared using CLARITY include:

  • Mouse brain
  • Human brain slices
  • Mouse spinal cord[3]

Comparison to Other Clearing Techniques

References

  1. K Chung, J Wallace, S-Y Kim, S Kalyanasundaram, AS Andalman, TJ Davidson, JJ Mirzabekov, KA Zalocusky, J Mattis, AK Denisin, S Pak, H Bernstein, C Ramakrishnan, L Grosenick, V Gradinaru, and K Deisseroth. Structural and molecular interrogation of intact biological systems. Nature (2013) 497: 332-337.
  2. K Chung and K Deisseroth. CLARITY for mapping the nervous system. Nature Methods (2013) 10(6): 508-513.
  3. M-D Zhang, G Tortoriello, B Hsueh, R Tomer, L Ye, N Mitsios, L Borgius, G Grant, O Kiehn, M Watanabe, M Uhlen, J Mulder, K Deisseroth, T Harkany, and TGM Hokfelt. Neuronal calcium-binding proteins 1/2 localize to dorsal root ganglia and excitatory spinal neurons and are regulated by nerve injury. PNAS (2014) 111(12): 1149-1158.