Background
- Lightsheet Background Information
- Successful 3D imaging of cleared biological samples with light sheet fluorescence microscopy
- Zeiss Lightsheet Sample Preparation
- Zeiss Quick Guide: Imaging with Water or Clearing Immersion
- Zeiss Improved Imaging of Cleared Samples
- Zeiss Insights into Tissue Clearing Imaging
- Adjusting Refractive Index for Clearing Applications
- Zeiss: The Art of Tissue Clearing
- The mesoSPIM Initiative
Reference Articles
- Clearing for Deep Tissue Imaging
- Tutorial: practical considerations for tissue clearing and imaging
- EZ Clear for simple, rapid, and robust mouse whole organ clearing
- Large-scale tissue clearing (PACT): Technical evaluation and new perspectives in immunofluorescence, histology, and ultrastructure
- Passive Clearing and 3D Lightsheet Imaging of the Intact and Injured Spinal Cord in Mice
- Practical Considerations for Quantitative Light Sheet Fluorescence Microscopy
- Tissue Clearing
- SeeDB: A Simple and Morphology-preserving Optical Clearing Agent for Neuronal Circuit Reconstruction
- Tissue clearing of both hard and soft tissue organs with the PEGASOS method
- Lightsheet Sample Processing - Embryonic Brain
- A hybrid open-top light-sheet microscope for versatile multi-scale imaging of cleared tissues - reviews a variety of clearing techniques
- Ethyl Cinnamate Based Clearing
Specimen Holders
- Overview of sample mounting options from the Huisken Lab.
- Protocol for viewing Zebrafish Embryos using light sheet microscopy.
Industry Resources
Please note the refractive index when researching these products.
- abcam - Tissue clearing made easy
- BioLegend - Ce3D Tissue Clearing
- Miltenyi Biotec - MACS Imaging Solution
- LifeCanvas technologies - Tissue clearing reagents
- Translucence Biosystems - 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 |