CLARITY Technique

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CLARITY (Clear Lipid-exchanged Anatomically Rigid Imaging/Immunostaining-compatible Tissue hYdrogel) is a technique developed in the Deisseroth lab at Stanford University.<ref>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.</ref><ref>K Chung and K Deisseroth. CLARITY for mapping the nervous system. ''Nature Methods'' (2013) 10(6): 508-513.</ref> The method is used to transform an intact tissue into an optically transparent and permeable hydrogel-hybridized form that can undergo immunostaining and 3-D imaging without damage to the sample.
 
CLARITY (Clear Lipid-exchanged Anatomically Rigid Imaging/Immunostaining-compatible Tissue hYdrogel) is a technique developed in the Deisseroth lab at Stanford University.<ref>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.</ref><ref>K Chung and K Deisseroth. CLARITY for mapping the nervous system. ''Nature Methods'' (2013) 10(6): 508-513.</ref> The method is used to transform an intact tissue into an optically transparent and permeable hydrogel-hybridized form that can undergo immunostaining and 3-D imaging without damage to the sample.
[[File: CLARITY_processing.png|thumb|CLARITY processing on a whole mouse brain]]
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[[File: CLARITY_processing.png|frame|CLARITY processing on a whole mouse brain]]
   
 
==Methodology==
 
==Methodology==

Revision as of 06:11, 19 April 2014

CLARITY (Clear Lipid-exchanged Anatomically Rigid Imaging/Immunostaining-compatible Tissue hYdrogel) 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 3-D imaging without damage to the sample.

(thumbnail)
CLARITY processing on a whole mouse brain

Contents

Methodology

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A schematic of the CLARITY process on a molecular level.

Biomolecules withing 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.

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 can successfully be 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.