CLARITY Tissue Clearing
An entire mouse brain made transparent — every neuron still in place, every synapse still legible, the whole organ glassy as a paperweight.
Renders intact tissue (a whole mouse brain, a thick slab of human cortex, a fly larva) optically transparent by infiltrating it with a hydrogel monomer mix, polymerising the gel in place, and then extracting the lipids. The result is a stable, water-rich tissue–gel hybrid that can be repeatedly stained with antibodies, dyes, or in-situ-hybridisation probes and imaged at single-cell resolution with light-sheet microscopy.
(1) Fixation and infiltration: tissue is perfused with a 4 % paraformaldehyde / 4 % acrylamide / 0.05 % bisacrylamide mixture and held at 4 °C overnight, allowing the monomers to diffuse throughout the tissue and crosslink to amine groups on proteins. (2) Polymerisation: the sample is degassed and warmed to 37 °C, triggering free-radical polymerisation that locks proteins, nucleic acids and small molecules into an acrylamide hydrogel covalently bonded to the tissue. (3) Lipid removal: the original Chung et al. 2013 protocol drove SDS micelles through the tissue with electrophoresis (active CLARITY) to extract membrane lipids in 1–7 days. Later passive variants (PACT, iDISCO, CUBIC, SHIELD) replace electrophoresis with simple incubation in detergent at 37–50 °C over 1–4 weeks. (4) Refractive-index matching: the cleared, lipid-free hydrogel is incubated in a high-refractive-index solution (FocusClear, RIMS, n ≈ 1.45) so the bulk RI matches that of fixed protein, eliminating scattering and producing transparency. (5) Labelling and imaging: antibodies, oligonucleotide probes, or fluorescent dyes diffuse through the gel and stain epitopes throughout the volume; light-sheet microscopy then images the entire intact organ at sub-µm in-plane resolution.
In plain terms
A brain is mostly fat, which is why it's opaque. CLARITY is like swapping all that fat for clear jelly. The neurons, the proteins, the wiring — all stay exactly where they were, just embedded in transparent gel instead of opaque cell membranes. Then you can shine a light right through the whole organ and watch every cell light up like fairy lights inside a jellyfish.
Also called: CLARITY, hydrogel-based clearing, passive CLARITY.
Why it matters
- Enables 3D reconstruction of long-range neuronal projections without sectioning, which always damages or loses inter-section connections.
- Compatible with antibody, RNA-FISH, and small-molecule labelling — proteins survive, nucleic acids survive, the tissue is multi-round-stainable.
- Foundation for many subsequent clearing protocols (iDISCO, CUBIC, SHIELD, MAP, ExLR) used in connectomics and pathology.
- Routinely combined with expansion microscopy and light-sheet microscopy to produce volumetric synaptic-resolution maps.
Where it lives
- Standard pipelines at the Allen Institute, Janelia, MIT, and most modern neuroscience labs
- LifeCanvas Technologies SmartClear/SmartSPIM commercial platform
- Stanford whole-organ CLARITY library (mouse brain atlas)
Key numbers
- Sample size: Up to whole adult mouse brain
- Hydrogel composition: 4 % acrylamide + 0.05 % BIS + 4 % PFA
- Refractive index after match: ~1.45
- Active clearing time: 1–7 days (Original 2013 protocol)
- Passive clearing time: 1–4 weeks (PACT, CUBIC variants)
Surprising facts
- An adult mouse brain (≈ 0.4 g) can be made fully transparent by CLARITY without cutting it — every region's neurons stay in their original three-dimensional positions.
- Once transparent, the same brain can be antibody-labelled, imaged, washed, and re-labelled with different markers many times — an experiment that destroys nothing.
- The technique was first published by Karl Deisseroth's group in Nature in 2013 and has since spawned more than a dozen daughter protocols collectively used in thousands of papers.
- CLARITY-cleared 5-mm-thick human prefrontal cortex slabs have been imaged at single-neuron resolution — the densest 3D neural reconstructions of human tissue ever obtained from light microscopy.
Open questions
- Inhomogeneous shrinkage and swelling: CLARITY-cleared tissue subtly shrinks and re-swells during washes; while average size is preserved within a few percent, local distortions on the scale of axonal trajectories are not zero and depend in poorly-characterised ways on tissue type and clearing protocol.
- Selective epitope loss: Some antibody epitopes survive CLARITY perfectly; others are stripped by the detergent or denatured by the hydrogel monomers. Predicting in advance which antibodies will work in cleared tissue remains largely empirical, despite multiple compatibility surveys.
A short history
CLARITY was developed by Kwanghun Chung, Karl Deisseroth and colleagues at Stanford, published in Nature in April 2013. The technique built on decades of clearing chemistry but was the first to combine hydrogel embedding with detergent extraction in a way that preserved structural integrity at whole-organ scale. PACT (Passive CLARITY Technique, Yang et al. 2014) eliminated the electrophoresis step. iDISCO (Renier et al. 2014, organic-solvent-based), CUBIC (Susaki et al. 2014, urea/Triton-based), SHIELD (Park et al. 2019, epoxide-based), and ExLR (expansion + light-sheet, 2020s) followed in rapid succession. As of 2026 a 'clearing' lab is a standard feature of mid-size and large neuroscience departments.
Sources
- Chung et al., Nature (2013) — original CLARITY paper (https://www.nature.com/articles/nature12107)
- Yang et al., Cell (2014) — PACT/PARS passive clearing (https://www.sciencedirect.com/science/article/pii/S0092867414009519)
- Park et al., Nat. Biotechnol. (2019) — SHIELD (https://www.nature.com/articles/s41587-018-0009-7)
- Tomer et al., Nat. Protocols (2014) — advanced CLARITY for fast, high-resolution mapping (https://www.nature.com/articles/nprot.2014.123)