Transparisation des échantillons biologiques
ImagoSeine offers a new service of rendering the biological samples optically transparent. We can adapt the clearing protocol to the biological sample and the corresponding labelling and propose the appropriate acquisition protocoles for the imaging.
Clearing of biological samples
In biological samples, light scattering and absorption are limiting factors for 3D imaging. Until now, thick biological samples were cut in thin layers to carry out 3D imaging. During the last decade, massive efforts were made to render sample transparent to light, by clearing methods for whole organ visualization.
The clearing methods are classified in two categories :
- Organic solvant based techniques.
The biological sample is dehydrated, delipidated and bleached (to remove pigments) and then the refractive index is matched using organic solvents. The most known tehniques are the BABB, 3DISCO and the iDISCO+. These methods are robust, highly recommended for big samples. They are however using toxic organic solvents, have some shrinkage side-effects on the sample and quench the fluorescence of fluorescent proteins.
- Hydrophylic reagent based techniques.
This category may be divided in 3 sub-categories :
- simple immersion.
Here the refractive index is matched by merging the sample in a medium (TDE, Fructose – SeeDB method, Glycerol). Easy method, works better for small samples, no shrinking, compatible with dyes, but cleraing not that efficient for big samples, and slow procedure.
Here the use of detergents removes the lipids and sucrose/urea causes hydration and clearing. Common techniques : CUBIC, Scale, Fruit. Efficient method, no quecnhing of fluorescent proteins, but mild expansion of the sample and quite slow.
- Hydrogel embedding.
Hydrogel empedding, and lipid removal with detergent or electrophoresis. The refractive index matching is achieved using immersion in TED, FocusClar, RIMS, Glycerol. Common techniques CLARITY, PACT/PARS, SWITCH. These methods are adapted to whole animal clearing, there is no fluorescent protein quenching, but are quite slow, and need special equipment.
- simple immersion.