Journal Club: advanced CLARITY and iDISCO papers

advanced CARITY
Cell paper, Yang et al (with Cai and Gardinaru, Deisserof former post-doc).

Recently developed clearing techniques

  • Clear T, Scale, CUBIC, SeeDB, CLARITY, passive CLARITY, advanced CLARITY, 3DISCO, i3DISCO etc
  • this paper, claims first whole organ imaging

Differences

  • different chemical
  • CLARITY: hydrogel and electrophoretic clearing + SDS
  • difficult and expensive to electrophoresis. Now have passive version, not ETC
  • 3DISCO uses THF/DBE. Faster, better clearing, high quenching of fluorescent proteins.
  • iDISCO – immuno labeling. Doesn’t suffer from FP quenching

Advanced CLARITY

  • passive clearing
  • index matched media

Optimization

  • screen different acrylamide and PFA and SDS for optimal concentrations
  • like 4% acrylamide + 0% PFA (brain expands a lot but is much clearer). increased detergent concentration.
  • also do FISH. much lower cellular background.
  • ‘RIMS’ reduce refractive index changes throughout the tissue. Change histodenz concentration
  • ‘PARS’ perfusion assisted (similar to cardiac perfusion fixation). Increases speed of clearing.
    • much less swelling (probably due to spatial confinement in body?)
    • do acralymide and fixation, and clearing during perfusion flow. (after prefusion fixation(?))

iDISCO technique

Paper info

  • rockefeller lab (also developer of 3DISCO)
  • also published in Cell last week

method

  • fix with 4% PFA
  • permiablize with methanol
  • immunolabel
  • tissue clearing THF/DCM/BM (DISCO)
  • image in DBE
  • imaged with Light-sheet microscopy
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