1. Gereral tissue clearing procedure

Tissue clearing technology has transformed the field of microscopy, enabling researchers to study whole organs and tissues in their complete 3D structure. With continued advancements in tissue clearing and light-sheet imaging methods, the scientific community anticipates gaining more insights into the complexities of biological systems, leading to new discoveries and breakthroughs.

The tissue clearing procedure involves several steps, including delipidation, decolorization, decalcification, 3D staining, and refractive index matching. During the process, lipids, pigments, and minerals are removed from the tissues, and their refractive indices are matched to the surrounding media to achieve light penetration, thus enabling imaging and analysis of complete structures.

Delipidation is a crucial step to achieving strong clearing results. Lipids have a higher refractive index than the surrounding biocomponents and form vesicles, leading to strong light scattering. Decolorization involves the removal of pigments that can interfere with imaging, while decalcification is an additional step added when bones or calcified cartilage are part of the sample.

Chemical dyes and antibodies are typically used to stain the tissue to highlight specific structures. However, staining probe penetration is occasionally problematic, and highly optimized protocols such as CUBIC-HistoVIsion, iDISCO, and eFLASH should be applied to overcome this issue.

Refractive index matching (RI matching) is a crucial step in tissue clearing to make the sample transparent while retaining its structural integrity. This is achieved by immersing the tissue in a solution with a high refractive index (RI = 1.45~1.55) that matches the refractive indices between the tissue and its surrounding medium.

While various protocols are available, it is essential to understand the basic clearing processes and their necessity to apply them appropriately to samples. Most modern clearing protocols are highly sophisticated and well-designed, leaving limited room for users to make significant modifications. Instead, researchers are encouraged to assess the advantages and disadvantages of each protocol and select the one that matches their research goals.

Selected literature:

Susaki and Ueda. Cell Chem Biol. (2016) 23:137

Tainaka et al. Annu Rev Cell Dev Biol. (2016) 32:713

Ueda et al. Nat Neurosci (2020) 21:61

Ueda et al. Neuron (2020) 106:369

Richardson et al. Nat Rev Methods Primer (2021) 1:84

for more intensive learning:

Handbook of Tissue Optical Clearing - New Prospects in Optical Imaging. Edited By Valery V. Tuchin, Dan Zhu, Elina A. Genina, Published February 9, 2022 by CRC Press. ISBN 9780367895099

2. Point to choose clearing protocols

Several tissue clearing protocols are available, each with its own advantages and disadvantages.

Organic solvent-based protocols, such as BABB, 3DISCO, and iDISCO, are widely used and achieve very strong tissue clearing efficiency, typically within a few days. However, these protocols require multiple reagent-exchanging procedures every several hours, and the samples are often shrunk. Compatibility with fluorescent proteins can occasionally be problematic, and organic compounds must be disposed of appropriately.
Some hydrophilic reagents, including CUBIC and MACS, also provide strong tissue clearing ability, and can be used to clear a broad range of tissue types and sizes. These protocols are safe and easy to use, with clearing typically requiring a relatively long treatment period (days to weeks). They are particularly designed to consider using fluorescence protein-labeled tissue samples, and sample size is occasionally swollen intending to increase clearing efficiency and imaging resolution. Combined protocols such as PEGASOS can also be applicable.
Tissue hydrogel chemistry, such as CLARITY and SHIELD, has emerged as another category of tissue clearing protocols. The hydrogel-based protocols also enable strong tissue clearing. In these protocols, the tissue is either embedded in an artificial gel or cross-linked with an epoxy compound, which gives durability for harsh physicochemical conditions (e.g., heat, SDS) and the potential to preserve biomolecules (e.g., RNA, metabolites). Therefore, they allow tissues to maintain their structural integrity. However, these protocols typically require the use of a specific device for the procedure, which can make initial parameter settings difficult.

When choosing a tissue clearing protocol, researchers must consider various factors such as the efficiency of clearing, sample types, compatibility with labeling tools, and imaging techniques, among others. For light-sheet imaging, in particular, a very strong clearing protocol should be chosen.

Selected literature:

Weiss et al. Nat Protoc. (2021) 16:2732

3. Why is CUBIC recommended?

CUBIC is one of the most recommended protocols for clearing tissues because it provides high-quality tissue clearing, is safe, and is easy to use. Additionally, samples can be embedded in agarose gel, which enables easy handling and prevents deformation during the clearing process. CUBIC is also compatible with the world-best 3D staining method, CUBIC-HistoVIsion, providing a remarkable opportunity for histological labeling of volumetric tissue samples.