What is Cleared-Sample Imaging?

Clearing-Sample/ Cleared-Tissue Imaging

Tissue clearing techniques have become a valuable tool for applications in 3D microstructure analysis of tissues (e.g. neuroscience, developmental biology, connectomics).

The different refractive indexes (RI) of the major components of biological tissue, i.e. water, lipids and proteins result in light scattering when light passes through the tissue. Tissue clearing modifies the optical properties of usually opaque samples to render them transparent while keeping their structure and fluorescent labels intact. After clearing, light can travel many millimeters through a specimen unrestricted from absorption and scattering, ideal for high-resolution microscopic imaging deep within the specimen.

Light-Sheet Microscopy leverages the optical advantages of cleared samples enabling fast, long-term, confocal-like optical sectioning and high-quality 3D imaging of cleared samples.

Methods for Optical Clearing

Tissue clearing methods homogenize the RI of a sample by removing, changing, or replacing some components.

Clearing methods can be grouped into two categories:

  • Solvent-based clearing methods (e.g. uDISCO, 3DISCO, BABB)
  • Aqueous-based clearing methods
    - Simple immersion (e.g. SeeDB, FAST-Clear)
    - Hyperhydration (e.g. CUBIC, ScaleS)
    - Hydrogel embedding (e.g. CLARITY, PACT/PARS)

No single clearing method will work for all tissue types, tissue sizes, and/or experiments.

Solvent-based clearing methods
Advantages: High-quality clearing, High-clearing speed, Long time storage of specimen
Disadvantages: Toxic and/or corrosive, Not suitable for lipid staining


Aqueous-based clearing methods
Advantages: Preservation of fluorescent protein emission, Preservation of lipids, Preservation of tissue architecture
Disadvantages: Slow clearing, Not suitable for big samples