Ramon y Cajal revolutionized biology by making visible what was previously invisible: individual brain cells and their interconnections. A recent Nature paper imparted the same excitement as Cajal’s illuminating treatise (Chung K, et al. Structural and molecular interrogation of intact biological systems. Nature. 2013;497(7449):332-337).1 Kai Deisseroth’s group overcomes major limitations of current techniques by inventing CLARITY, a transformative method that enables 3-dimensional structural and molecular analyses of topologically intact brains and other biological tissues.
The authors postulated that non-destructive removal of lipid membrane bilayers that make tissues impermeable to macromolecules and photons would enable whole organ imaging. They hypothesized that maintaining the tissue’s structural scaffolding and integrity would best preserve an organ’s intrinsic biological information. CLARITY converts intact tissues and organs into optically transparent, macromolecule-permeable structures, thus providing a natural anatomical unit that is conserved for molecular and structural analysis.
To maintain tissue morphology, the authors infused hydrogel monomers, formaldehyde, and thermally triggered initiators into tissues at 4°C. Formaldehyde caused tissue crosslinking and provided covalent linkage of the hydrogel monomers to cellular proteins, nucleic acids and small molecules. Then a tissue-hydrogel hybrid construct was created via 37°C incubation of infused tissues. This hybridization provided tissue support and chemical incorporation of biomolecules into the hydrogel mesh. Unbound lipids and other biomolecules were then rinsed away with a novel ionic detergent extraction that does not quench fluorescence, a common problem of currently used organic lipid extraction solvents. The authors also created a technique called electrophoretic tissue clearing (ETC) that uses charged ionic micelles to create a tissue active-transport system accelerating detergent micelle diffusion into tissues.
To demonstrate CLARITY technology, the authors completely processed a whole mouse brain in 8 days. The intact transparent brain was imaged in a refractive-index-specific solution matching the CLARITY hybrid, to prevent scattering from macromolecular heterogeneity of the protein and nucleic acid complexes. Therefore, whole mouse brain imaging at cellular resolutions was achieved even with single-photon microscopy (Figure). Researchers viewed fluorescence-labeled neurons in areas as deep as the thalamus, limited only by the microscope objective’s working distance.
The authors demonstrated CLARITY’s ability to provide molecular phenotyping in addition to structural information. Native antigens are preserved in the hydrogel-hybridization process. CLARITY processing resulted in far less protein loss compared to traditional methods. Immunohistochemistry on intact mouse brains was successful, and only moderate signal attenuation was observed in the deepest brain regions even after 2 weeks of incubation. Furthermore, small biomolecules such as GABA and messenger RNAs were also preserved. CLARITY-processed brains were able to withstand multiple rounds of elution without antigen degradation, thus demonstrating another advantage over traditional elution methods that cause antibody loss and fluorescence quenching.
Chung et al further demonstrated that CLARITY technology is applicable to archived human tissues. They used CLARITY to process post-mortem human brains, and successfully performed immunohistological visualization and identification of neuronal projections. Formalin-fixed frontal lobe tissues from autism patients (stored for more than 6 years) were successfully stained with neuro-filament protein and myelin basic protein to trace individual axonal fibers. They further visualized the distribution of parvalbumin-labeled neurons and processes in these autistic brains, and discovered that many parvalbumin+ interneuron connections formed ‘ladder-like’ abnormalities previously observed with mutant Down-syndrome cell-adhesion molecule (Dscam) or protocadherin proteins. Previously unknown, this link between abnormal parvalbumin+ neuron connections and genes associated with autism-related disorders revealed through CLARITY molecular/functional analysis exemplifies the potential of this technique.
CLARITY technology has broad clinical implications. For example, CLARITY will allow researchers and clinicians to view normal neuronal networks and trace detailed changes associated with various disease processes. Large clinical banks of rare disease-associated tissues can be examined after CLARITY processing. Due to the stable hydrogel-tissue hybrid, these precious, rare samples will also remain available and reusable for many tests. CLARITY provides a novel method to preserve and examine intact brains and other tissues for structural and molecular relationships, and better define normal and diseased states of the intricate human brain.
1. Chung K, Wallace J, Kim SY, et al.. Structural and molecular interrogation of intact biological systems. Nature. 2013;497(7449):332–337.