In the March issue of Nature Methods, the technology feature explores some ways that labs are optimizing probes to image cleared tissue. As we interviewed scientists, we learned about published work and ongoing unpublished experiences. Here is a snapshot of how some probes work with some clearing methods. It’s an imperfect table and is likely to evolve as research continues. We welcome your comments. We know there are different viewpoints and varying experiences and we hope it will be helpful to others to hear about them.
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|Probe||Labeling shown in large samplesb||Labeling shown in small samples||Does not work well?|
|Genetically encoded fluorescent proteins||3DISCO (signal quenched after a few days), CLARITY, CUBIC, ExM (variant)a, PACT/PARS (signal retained 6 months and longer)a, sDISCOa||
BABB (signal quenched after a few hours), ClearT2, ExM (variant)a , ExM/ePACTa , ScaleA2, ScaleS, SeeDB,
|Immunolabels||3DISCO, CLARITY, iDISCO, iDISCO+a, iSeeDB, PACT/PARS, Sucrose, SWITCH||BABB, ClearT, ClearT2, CUBIC, SeeDB, SeeDB2a (in press) Sucrose, TDE, ExM|
|Specific nucleic acid detection||EDC-CLARITY||CLARITY, ExM (variant)a, PACT/PARS|
|Dyes and stains|
|Lipophilic dyes such as DiI, Sudan Black||PACT/PARS (Sudan Black), SWITCH (DiI)||ClearT, ScaleS, SeeDB|
|Nuclear stains such as DAPI, DRAQ5, SYTO and PI||CUBIC, ExM (variant)a, PACT/PARS, SWITCH||SeeDB, SeeDB2a|
|SNAP-tags with SiR probes||PACT/PARS, BABB, Scale||SeeDB|
Clearing method: Solvent-based; simple immersion; hyperhydration; hydrogel-based.
bIndicates larger samples such as whole organs
Sources: E. Boyden, MIT; K. Chung, MIT, K. Deisseroth, Stanford University; H-U. Dodt, Vienna University of Technology/Medical University of Vienna; V. Gradinaru, Caltech; P. Heppenstall, EMBL; Takeshi Imai, RIKEN; K. Johnsson, EPFL; J. Lichtman, D. Richardson, Harvard University; A. Miyawaki, RIKEN; M. Tessier-Lavigne, N. Renier, Rockefeller University.
Glossary of some tissue clearing agent acronyms
3DISCO : Three-dimensional imaging of solvent-cleared organs
sDISCO: Stabilized three-dimensional imaging of solvent-cleared organs
BABB : Benzyl alcohol and benzyl-benzoate
CLARITY : Clear Lipid-exchanged Acrylamide-hybridized Rigid Imaging/Immunostaining/In situ hybridization-compatible Tissue-hYdrogel
CUBIC : Clear unobstructed brain imaging cocktails and computational analysis
EDC-CLARITY : 1-Ethyl-3-3-dimethyl-aminopropyl carbodiimide-CLARITY
ePACT: PACT-based expansion clearing.
iDISCO : Immunolabeling-enabled 3D imaging of solvent-cleared organs
iDISCO+ : Immunolabeling-enabled 3D imaging of solvent-cleared organs plus
PACT : Passive Clear Lipid-exchanged Acrylamide-hybridized Rigid Imaging/Immunostaining/In situ hybridization-compatible Tissue-hYdrogel
PARS : Perfusion-assisted agent release in situ
SeeDB : See Deep Brain
Spalteholz’s preparation : Benzylbanzoate/methylsalicate
TDE : 2,2'-thiodiethanol
Some lab resource pages
Chung lab resources – Literature, protocols, videos and discussion pages from the MIT lab of Kwanghun Chung related to SWITCH, electrostochastic transport and CLARITY.
Expansion microscopy resources – Literature and protocols related to expansion microscopy developed in the MIT lab of Edward Boyden.
iDISCO resources – Literature, protocols and information about validated antibodies related to iDISCO
SeeDB Resources – Protocols, literature videos related to SeeDB developed in the RIKEN lab of Takeshi Imai.