Background
- Lightsheet Background Information (opens in a new tab)
- Successful 3D imaging of cleared biological samples with light sheet fluorescence microscopy (opens in a new tab)
- Zeiss Lightsheet Sample Preparation (opens in a new tab)
- Zeiss Quick Guide: Imaging with Water or Clearing Immersion (opens in a new tab)
- Zeiss Improved Imaging of Cleared Samples (opens in a new tab)
- Zeiss Insights into Tissue Clearing Imaging (opens in a new tab)
- Adjusting Refractive Index for Clearing Applications (opens in a new tab)
- Zeiss: The Art of Tissue Clearing (opens in a new tab)
- The mesoSPIM Initiative (opens in a new tab)
Reference Articles
- Clearing for Deep Tissue Imaging (opens in a new tab)
- Tutorial: practical considerations for tissue clearing and imaging (opens in a new tab)
- EZ Clear for simple, rapid, and robust mouse whole organ clearing (opens in a new tab)
- Large-scale tissue clearing (PACT): Technical evaluation and new perspectives in immunofluorescence, histology, and ultrastructure (opens in a new tab)
- Passive Clearing and 3D Lightsheet Imaging of the Intact and Injured Spinal Cord in Mice (opens in a new tab)
- Practical Considerations for Quantitative Light Sheet Fluorescence Microscopy (opens in a new tab)
- Tissue Clearing (opens in a new tab)
- SeeDB: A Simple and Morphology-preserving Optical Clearing Agent for Neuronal Circuit Reconstruction (opens in a new tab)
- Tissue clearing of both hard and soft tissue organs with the PEGASOS method (opens in a new tab)
- Lightsheet Sample Processing - Embryonic Brain (opens in a new tab)
- A hybrid open-top light-sheet microscope for versatile multi-scale imaging of cleared tissues (opens in a new tab) - reviews a variety of clearing techniques
- Ethyl Cinnamate Based Clearing
Specimen Holders
- Overview of sample mounting options from the Huisken Lab (opens in a new tab).
- Protocol for viewing Zebrafish Embryos using light sheet microscopy (opens in a new tab).
Industry Resources
Please note the refractive index when researching these products.
- abcam (opens in a new tab)- Tissue clearing made easy
- BioLegend (opens in a new tab)- Ce3D Tissue Clearing
- Miltenyi Biotec - MACS Imaging Solution
- LifeCanvas technologies (opens in a new tab) - Tissue clearing reagents
- Translucence Biosystems (opens in a new tab) - Universal tissue clearing kit
Clearing Methods
| Method | Author | RI | Components | LS Z.1 | Commnent |
|---|---|---|---|---|---|
| Scale A,B,U | Miyawaki, Riken, JP | 1.38 | Glycerol, Urea, Triton | + | Alternative to BABB, less quenching, for myelinated tissue |
| CLARITY | Chung and Deisseroth, Stanford, USA | ~1.45 | SDS PAGE and electrophoretic clearing | ++ | Very good results when it works, high failure rate (> 50 %) |
| CUBIC | Susaka et al, Riken, JP | 1.47- 1.49 | Optimized Scale | ++ | Published in Cell, April 2014 |
| FocusClear | CelExplorer Labs | ~1.45 | Commercial medium | ++ | |
| RapiClear | CelExplorer Labs | 1.47 – 1.55 | Based on FocusClear, more flexible in RI, cheaper | + | Comes in variants 1.47 – 1.49 – 1.52 - 155 |
| TDE | Hell et al, various labs tried | Tunable | 97% 2,2’-Thiodiethanol in water | + | TDE as mounting medium for different clearing (e.g. DMSO) , difficulties in reproducibility |
| LUMOS | Efimova et al, Moscow | 1.45 - 1.47 | Not Known | ++ | Good results, some scattering left Compatible with immunostainings, in situs and lipophilic tracer dyes |
| Glycerol/H2O (80/20) | Generic | 1.45 | Simple mix of Glycerol and Water | + | Easy to reproduce, cheap |
| PACT, RIMS | Bin Yang et al. | 1.45 | Perfusion assisted clearing, | + | Lab of Viviane Gradinaru |
Clearing Methods - Organic Solvent Based
The following clearing methods are organic solvent based and are not directly compatible with the Lightsheet Z.1 because they may damage the optical components. Samples may be protected by enclosing them in glass (Simax, refractive index 1.472), which enables use with a lightsheet system. See the associated white paper for expanding Simax-compatible clearing solutions when working with these organic solvents. (T. Pratt, Zeiss.)
| Method | Author | Refractive Index (RI) | Components | Comments |
|---|---|---|---|---|
| SeeDB | Ke et al, JP | 1.47- 1.49 | Sucrose / Fructose saturated | Very high viscosity sugar solutions, hard to get homogeneous medium, buoyancy of specimen, bubbles. |
| ClearT, ClearT2 | Kuwajima et al, 2013 | ~1.45 | formamide/ polyethylene glycol | Formamide is highly corrosive, will damage dipping objectives |
| BABB | Spalteholz 1914 | 1.53-1.57 | Benzyl alcohol - Benzyl Benzoate | Aka Murray’s Clear, LSFM: Dodt, Stelzer, Giese |
| Spalteholz | Spalteholz 1914 | 1.54 | Commercial medium | |
| 3DISCO | Erturk et al | 1.56 | developed starting from BABB; THF, DBE, DCM | Fast and cheap clearing, fast fluorescent quenching |