TABLE 1.
Key features of the main tissue processing methods discussed.
| Protocol | Fixation/Cross-linking | Permeabilization and Delipidation | Decolorization | Preservation of fluorescence from FP | Labeling/Staining | Clearing performance [RI], Reagent | Effect on tissue size | Max. # Resolution | Clearing time†‡¶ | Application to neuropathology Related computational tools |
| Hydrophobic (Organic Solvent) Tissue Clearing | ||||||||||
| BABB (Dodt et al., 2007) | Standard PFA perfusion | Strong: EtOH, DCM | - | Low (hours) | Thioflavin-S for Aβ plaques and lectin for vasculature (Huang et al., 2015). | Very high [1.56], BABB | Slight shrinkage | 0.5-2 μm | 7 days† | Visualization of Aβ plaques and vasculature in entire mouse brains (Huang et al., 2015). |
| 3DISCO (Ertürk et al., 2012a, b) | Standard PFA perfusion | Strong: THF, DCM | - | Low (hours) | - | Highest [1.56], BABB | Shrinkage (∼30% volume) | 0.5-2 μm | 2 days† | Assessment of axonal degeneration and regeneration in spinal cord injury (Ertürk et al., 2012b). |
| iDISCO (Renier et al., 2014) iDISCO + (Renier et al., 2016) https://idisco.info/ | Standard PFA perfusion | Strong: Methanol, DCM | H2O2 | Low (hours) | Antibodies, Congo red for Aβ plaques | Highest [1.56], DBE | Minimal | 0.5-2 μm | 7-14 days† | Evaluation of Aβ pathology in AD transgenic mice and human brains; automated quantification of Aβ plaques with ClearMAP (Liebmann et al., 2016). Imaging of temporal/spatial progression of tau neuropathology in EC-tau mice (Fu et al., 2016). |
| u/vDISCO (Pan et al., 2016) (Cai et al., 2019) discotechnologies.org | Standard PFA perfusion | Strong: tert-Butanol, THF DCM/Triton-X100 | Aminoalcohol | Acceptable (hours-days) | Nanobodies (vDISCO; 2-3 d. active or 5-7 d. passive staining) | Highest [1.56], BABB-D | Shrinkage (∼35–55% volume) | 1-2 μm | 5 days† | Machine learning pipeline for identification and quantification of nanobody-labeled cells (Pan et al., 2019). |
| SHANEL* (Zhao et al., 2020) discotechnologies.org | PFA perfusion of entire human organs | Strong: CHAPS, DCM | CHAPS/NMDEA | N/A | Antibodies, Methoxy-X04 for labeling Aβ plaques | Very high [1.56], BABB | Shrinkage (∼30–45% volume) | 1-2 μm | 1- > 3‡weeks | Detection of amyloid plaques in human brain tissue (POC); deep learning-based pipeline for 3D reconstruction and data analysis (Zhao et al., 2020). |
| Hydrophilic Tissue Clearing | ||||||||||
| CUBIC 1/2 (Susaki et al., 2014) cubic.riken.jp | Standard PFA perfusion | Strong: ScaleCUBIC-1 (15% Triton-X100 + 25% Aminoalcohol + 25% Urea) | Aminoalcohol | High | Antibodies | High [1.49], ScaleCUBIC-2 (urea + aminoalcohol + sucrose) | Slight expansion | 0.5-2 μm | 7-14 days† | Visualization of detailed neuronal morphology and Aβ plaques using Golgi and Thioflavin-S staining in AD mice (POC) (Vints et al., 2019). |
| Updated CUBIC protocols (Tainaka et al., 2018) cubic.riken.jp | Standard PFA perfusion | Strong: CUBIC-L (10% Triton-X100 + 10% Aminoalcohol) | Aminoalcohol | High | Antibodies | High [1.52], CUBIC-R/RA (antipyrine, nicotinamide) | Slight expansion | 0.5–2 μm | 5–11 days† | – |
| ScaleS* (Hama et al., 2015). | Standard PFA perfusion | Mild: urea + sorbitol + DMSO | – | High | Antibodies, Lectin | High [1.44] (urea + sorbitol + DMSO) | Minimal | 0.5–2 μm | 2–5 days for mm-thick slides. | Evaluation of Aβ pathology, microglia and EM ultrastructure in AD transgenic mice and human AD brains (Hama et al., 2015). |
| Hydrogel-Based Tissue Clearing | ||||||||||
| CLARITY (Chung et al., 2013; Tomer et al., 2014) clarityresourcecenter. com | PFA/acrylamide/bis-acrylamide | Strong: 4% SDS | – | Temperature-dependent: preserved at 37°C | Antibodies | Good [1.45], FocusClear | Minimal | 0.5–2 μm | 1 (active)-3 (passive) weeks† | Visualization of Aβ plaques and tangles in human AD brain (POC) (Ando et al., 2014). Identification of fragmented nigrostriatal axons in PD mice (Nordström et al., 2015). Visualization of Lewy pathology in human brain (POC) (Liu et al., 2016). |
| SWITCH* (Murray et al., 2015) chunglabresources.com | PFA, Glutaraldehyde | 200 mM SDS | – | Temperature-dependent: preserved at 37°C requiring longer clearing | Antibodies, Lectin | High, [1.47], diatrizoic acid, n-methyl-D-glucamine, iodixanol | Minimal | 0.5–2 μm | 2–4 weeks† | Assessment of Aβ deposits in 5xFAD mice, revealing early Aβ accumulation in subcortical areas (Gail Canter et al., 2019). |
| SHIELD* (Park et al., 2019) chunglabresources.com | PFA, Polyepoxide | 300 mM SDS | - | Time/temperature-dependent: preserved at 45°C | Antibodies, FISH, Lectin | High, [1.46], Iodixanol- or Iohexol-based Protos media | Minimal | 0.5–2 μm | 1 (active) -4 (passive) weeks† | Quantitative evaluation of Aβ pathology in 5xFAD mice exposed to gamma sensory stimulation (Martorell et al., 2019). |
| Tissue Expansion | ||||||||||
| ExM/ProExM/iExM (Chen et al., 2015; Chang et al., 2017; Asano et al., 2018) expansionmicros copy.org | PFA, acrylamide/bis-acrylamide/sodium acrylate Acryloyl-X | 5% Triton X-100, 1% SDS (for post expansion staining) | – | Partial, only digestion-resistant FP | Antibodies, FISH | High, [∼1.33], (water) | 20 × (iExM) 4.5 × (ExM) Expansion (3.5 × for FISH) | ∼25 nm (iExM) or 60 nm (ExM) | 1-2 days ¶ | Identification of perivascular c43 accumulation on astrocytic endfeet in human brain epilepsy (Deshpande et al., 2017). Identification of eRF1 accumulation within nuclear envelope invaginations in iPSC-derived neurons from ALS patients (Ortega et al., 2020). |
| ExPath* (Zhao et al., 2017; Bucur et al., 2020) | PFA, formalin or acetone. Acrylamide/bis-acrylamide/sodium acrylate | 0.1% Triton X-100 | – | N/A | Antibodies, DNA-FISH | High, [∼1.33], (water) | 4.5× | ∼60 nm | 4 h (≤5 μm) – 2 days ¶ | – |
| MAP (Ku et al., 2016) chunglabresources.com | PFA, acrylamide/bis-acrylamide/sodium acrylate | 200 mM SDS | – | Fluorescence is lost but epitopes are preserved | Antibodies | [1.47] pre-expansion. [∼1.33] (water) after expansion. | Fourfold linear expansion | ∼60-nm | ∼1 week ¶ | – |
| X10 ExpM (Truckenbrodt et al., 2018, 2019) | Sodium acrylate, N,N-dimethyl acrylamide, Acryloyl-X | 0.1% Triton X-100 | – | Fluorescence is lost but epitopes are preserved | Antibodies | High, [∼1.33], (water) | Tenfold expansion | ∼25 nm | 3 days ¶ | – |
| SHIELD-MAP (3x linear expansion) (Park et al., 2019) chunglabresources.com | PFA, Polyepoxide acrylamide/bis-acrylamide/sodium acrylate/VA-044 | 300 mM SDS | – | Time/temperature-dependent. 27-fold signal reduction after expansion. | Antibodies | DI water for expansion. | Threefold linear expansion | ? | ∼2 weeks ¶ (1–3 mm Slices) | – |
#Lateral resolution using standard (diffraction-limited) light microscopy. *Optimized for human tissue. †For an intact mouse brain. ‡For ∼1 cm3 human brain tissue. ¶Expansion of brain slices. DCM, dichloromethane. CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate. BABB, benzyl alcohol + benzyl benzoate (1:2). BABB-D, BABB + Dibenzyl ether. SDS, sodium dodecyl sulfate. POC, proof-of-concept application.