CLARITY

CLARITY
Name	CLARITY
Classification	Tissue clearing, Neuroimaging
Inventor	Kwanghun Chung, Karl Deisseroth
Institution	Stanford University, MIT
Year	2013
Related	PACT, PARS, CUBIC, iDISCO

CLARITY is a transformative tissue clearing technique in biomedical research that renders biological specimens optically transparent while preserving their molecular and structural integrity. Developed primarily for the intricate study of the nervous system, it enables high-resolution, three-dimensional imaging of entire organs without the need for physical sectioning. The method represents a significant leap in connectomics and systems neuroscience, allowing researchers to visualize complex neural networks within intact brains and other tissues.

Definition and Overview

CLARITY, an acronym for **C**lear **L**ipid-exchanged **A**crylamide-hybridized **R**igid **I**mmunolabeling-enabled **T**issue-h**Y**drogel, is a chemical process that converts biological tissue into a nanoporous hydrogel-hybrid form. This foundational innovation allows for the removal of light-scattering lipids, which are the primary source of opacity in tissues, while retaining proteins and nucleic acids in their native spatial configuration. The technique facilitates deep-tissue imaging using modalities like confocal microscopy and light-sheet fluorescence microscopy, providing unprecedented views of cellular architecture. Its development was a collaborative effort between the laboratories of Karl Deisseroth at Stanford University and Kwanghun Chung, then at Stanford University and later at the Massachusetts Institute of Technology.

Historical Development

The pursuit of tissue transparency has a long history in histology, with early methods like Spalteholz's clearing using organic solvents. Modern neuroscience's drive to map the connectome of model organisms like the mouse created a pressing need for intact-tissue imaging. Prior to CLARITY, techniques such as BABB and 3DISCO offered clearing but often compromised fluorescence or caused tissue distortion. The pivotal work was published in the journal *Nature* in 2013 by the team led by Kwanghun Chung and Karl Deisseroth, demonstrating the first fully hydrogel-embedded, lipid-cleared mouse brain. This breakthrough quickly influenced fields beyond neuroscience, including developmental biology and oncology.

Methodological Process

The CLARITY protocol involves several critical steps. First, tissue is perfused and fixed with formaldehyde and then infused with acrylamide monomers and a thermal initiator like VA-044. The sample is then heated to form a hydrogel mesh that covalently binds to biomolecules. The embedded tissue is placed in an electrophoretic chamber with a SDS buffer, and an electric field actively pulls out the lipids in a process termed electrophoretic tissue clearing. Alternatively, a passive diffusion method can be used. After clearing, the transparent hydrogel-tissue hybrid can be stored in a refractive-index matching solution like FocusClear or RIMS. It is then amenable to multiple rounds of staining with antibodies or nucleic acid dyes for specific molecular labeling.

Applications in Neuroscience

CLARITY has become a cornerstone tool for investigating the architecture of the central nervous system. Researchers have used it to trace long-range axonal projections across entire rodent brains, map the distribution of specific neuronal types defined by markers like parvalbumin, and study pathological protein aggregates in models of Alzheimer's disease and Parkinson's disease. Landmark projects, such as the Mouse Light Project at the Allen Institute for Brain Science, have utilized CLARITY-based methods to create comprehensive maps of neuronal connectivity. It has also been applied to human brain tissue samples from brain banks to study post-mortem neuroanatomy in health and disease.

Advantages and Limitations

The primary advantage of CLARITY is its ability to preserve endogenous fluorescence and enable robust immunolabeling in a structurally intact, transparent sample, allowing for phenotyping of cells in their full three-dimensional context. However, the technique has limitations. The original active clearing process requires specialized equipment and can be time-consuming, taking weeks for whole adult mouse brains. The hydrogel embedding can also cause slight tissue expansion, and very dense or myelinated tissues can remain challenging to clear completely. Furthermore, the depth of antibody penetration for labeling large samples can be inconsistent, though advancements like SWITCH have addressed some of these issues.

Related Techniques

The success of CLARITY spurred rapid innovation in the field of tissue clearing, leading to numerous alternative and optimized protocols. These include PACT and PARS, which are passive clearing methods also developed by Kwanghun Chung; CUBIC, developed by Hiroki R. Ueda's team in Japan, which is particularly effective for decolorization; and iDISCO, optimized for immunolabeling of large samples. Other notable methods are ScaleS, SeeDB, and BABB-based techniques. Each method offers different trade-offs in speed, compatibility with specific labels, and preservation of tissue morphology, creating a versatile toolkit for researchers across biology.

Category:Laboratory techniques Category:Neuroimaging Category:Histology