CLARITY is a tissue-clearing technique that turns opaque biological tissue, including entire mouse brains, into a transparent structure that can be imaged in three dimensions without ever slicing it. Developed in 2013, the method works by replacing the lipids that scatter light with a transparent hydrogel scaffold, while preserving proteins, nucleic acids, and the physical architecture of the tissue. The result is something that looks almost like a glass organ but still contains its original molecular information, allowing researchers to trace neural circuits, map cell populations, and stain the same sample repeatedly. What makes it especially powerful for neuroscience is the ability to follow a single neuron’s wiring across an intact brain, a task that traditional thin-section microscopy handles poorly.
Turning Brain Tissue Transparent
The core idea behind CLARITY is replacing the components of tissue that block light while leaving everything else in place. In a living brain, cell membranes and myelin sheaths are packed with lipids, and these lipids are the main reason brain tissue is opaque. They scatter photons in every direction, which is why you cannot see through even a thin slice of fresh brain. CLARITY removes those lipids but first locks all the other important molecules into a supportive mesh so nothing falls apart.
The process starts by infusing the tissue with a mixture of hydrogel monomers and formaldehyde. The formaldehyde crosslinks proteins and nucleic acids to the hydrogel monomers, anchoring them in place. When the mixture is heated, it polymerizes into a mesh that runs through the entire tissue, physically holding the biological structures together. At this point, the tissue is still opaque because the lipids remain. The next step is to wash those lipids out using a detergent solution. Once the lipids are gone, the tissue becomes a nanoporous hydrogel-tissue hybrid that is structurally intact but allows light to pass through. The tissue also becomes permeable to large molecules like antibodies, which can now diffuse deep inside for staining.
Active Versus Passive Lipid Removal
The original 2013 protocol used electrophoresis to speed up the lipid-removal step. An electric field drives the detergent micelles through the tissue, pulling lipids out faster than simple soaking would. This electrophoretic tissue clearing can render a whole mouse brain transparent in days rather than weeks. But it comes with practical headaches: the equipment is specialized, the electric current can damage tissue if not carefully controlled, and the setup is finicky enough that many labs struggled to replicate it consistently.
That led to the development of passive CLARITY, which skips the electrophoresis entirely and relies on the detergent diffusing through tissue on its own, typically at elevated temperature. Passive clearing is slower, often taking weeks for a whole mouse brain, but it is far simpler and causes less tissue damage. One widely used passive protocol demonstrated that combining passive clearing with an inexpensive refractive-index matching solution called TDE (2,2′-thiodiethanol) could produce good optical transparency at a fraction of the cost of the original method.
Optimization work found that for whole mouse brains, electrophoresis was sometimes necessary to achieve full transparency, with one systematic study reporting that optimal clearing required about five days of electrophoresis using a combination of 37°C and 55°C temperatures. For smaller tissue blocks or less dense organs, passive clearing often works well enough, making the technique accessible to labs without electrophoresis rigs.
Making Cleared Tissue Optically Useful
Removing lipids alone does not make tissue perfectly transparent. Light still bends when it passes between materials with different refractive indices, the way a straw looks bent in a glass of water. After clearing, the hydrogel-tissue matrix has a refractive index around 1.45, and the surrounding liquid needs to match that number closely or the tissue will remain hazy. This step, called refractive-index matching, turns out to be one of the trickiest parts of the whole workflow.
The original protocol recommended a proprietary product called FocusClear, which matched the refractive index well but was expensive enough to be prohibitive for routine use. Researchers quickly found cheaper alternatives. TDE emerged as a popular option because its refractive index can be tuned simply by adjusting the concentration: a 47% TDE solution matches typical microscope objectives with a refractive index around 1.42, while a 63% solution matches FocusClear’s 1.45. Studies confirmed that 47% TDE preserved mouse brains and produced uniform transparency comparable to the proprietary reagent.
The choice of matching solution is not one-size-fits-all, though. Work on human brain tissue found that solutions performing well in mouse tissue sometimes failed on human samples, which tend to be denser and more heavily fixed. One group reported that a commonly recommended alternative called Histodenz worked for transgenic mouse tissue but not for human brain, highlighting that the clearing and matching protocols often need to be tuned for each tissue type. They also discovered that the solvent used to dilute TDE matters: switching from phosphate-buffered saline to plain phosphate buffer reduced unwanted yellowing and improved transparency, though using water instead could damage fluorescent signals through pH shifts.
Getting Antibodies Deep Inside
Transparency is only half the story. The real payoff of CLARITY is being able to label specific cell types and molecules inside the intact tissue and then image them in three dimensions. In traditional histology, you slice tissue into sections a few micrometers thick, stain each slice, and image them individually. With CLARITY, you can stain the whole volume at once and image straight through it. But getting antibodies to penetrate centimeters of tissue is a challenge, even when the tissue is porous.
Antibodies are large proteins, and their diffusion through even cleared tissue is slow. In a standard mouse brain, complete antibody penetration can take days. One group developed a modification using boric acid buffer at pH 7.2, which expanded the hydrogel pores and significantly improved antibody penetration. In their tests, antibody penetration depth after a few hours of centrifugation reached about 180 micrometers in boric acid buffer compared to 133 micrometers in standard buffer. More importantly, this approach cut the staining time for a whole intact mouse brain from roughly 40 hours per antibody to about 24 hours.
For human brain tissue, where samples can be much thicker and denser, the process takes considerably longer. A pilot study achieved immunohistochemical staining of human post-mortem brain tissue up to five millimeters thick, using a combination of active and passive clearing with extended staining times. That kind of depth is remarkable for intact tissue staining, but it underscores that scaling CLARITY to human-sized samples remains a slow and labor-intensive process.
Imaging at Scale With Light-Sheet Microscopy
A transparent brain is only useful if you can image it quickly and at high resolution. Conventional confocal microscopes scan one point at a time, which means imaging a whole mouse brain at cellular resolution could take weeks of continuous scanning. Light-sheet microscopy changed the game by illuminating an entire plane of tissue at once, collecting images hundreds of times faster.
CLARITY-optimized light-sheet microscopy, or COLM, was specifically designed to work with cleared tissue. The approach pairs optimized objectives with light-sheet optics to accelerate data collection by several orders of magnitude compared to point-scanning methods, while maintaining or even improving resolution. This makes it practical to image an entire clarified mouse brain at cellular resolution in hours rather than months.
The datasets that come out of these imaging sessions are enormous. A single cleared and imaged mouse brain can produce terabytes of raw image data, thousands of individual image tiles that need to be stitched together, corrected for artifacts, and aligned to a standard brain atlas. Dedicated computational pipelines have been developed to handle this, using parallel processing to stitch tiles into a single volumetric image and align the result to reference atlases. Software tools like BigStitcher and Terastitcher-based pipelines handle the stitching, while atlas alignment lets researchers compare results across different animals. Without these computational tools, the imaging data would be essentially unusable.
Beyond the Mouse Brain
CLARITY was developed with neuroscience in mind, but the same principle works on many other tissues. Researchers have adapted the technique to clear and image intact kidneys, lungs, livers, pancreases, and intestines. A related variant called PARS (perfusion-assisted agent release in situ) takes the concept even further by pumping clearing reagents through an animal’s own circulatory system, clearing entire bodies in place. Using PARS, researchers achieved complete clearing of all major organs within about a week, and cleared the central nervous system within two weeks, in both mice and rats.
The technique has also been adapted across species. A protocol called zPACT was developed specifically for zebrafish, a commonly used model organism in developmental biology. The method renders juvenile zebrafish specimens optically transparent while preserving tissue architecture, and has been successfully tested on other aquatic species including medaka fish and Xenopus frogs. These cross-species adaptations show that the basic chemistry of hydrogel embedding and lipid removal is flexible enough to work across a range of tissue types, though each new species or organ typically requires protocol adjustments.
Applications in Human Disease
Some of the most compelling uses of CLARITY involve human tissue from patients with neurological disease. In Parkinson’s disease, for example, researchers used the technique to visualize Lewy bodies, the hallmark protein aggregates of the disease, in three dimensions within intact human brain tissue. Traditional thin-section histology shows Lewy bodies as flat circles on a slide, but CLARITY revealed their full three-dimensional morphology and distribution within neural circuits, offering a richer picture of how the pathology spreads.
Cancer research has also adopted the approach. Studies on human breast cancer tissue demonstrated that core needle biopsy samples could be processed through the CLARITY workflow, with cellular morphology preserved well enough that the technique could potentially be integrated into clinical pathology. The ability to see tumor architecture in three dimensions, rather than inferring it from a handful of two-dimensional slices, could change how pathologists assess margins and tumor heterogeneity.
For research on the aging human brain, CLARITY has been applied to post-mortem tissue from elderly donors, including a study that used brain samples from a 99-year-old subject stored in formalin for six months. Working with archival tissue like this is important because brain banks contain decades’ worth of preserved samples from patients with well-characterized clinical histories. If CLARITY can extract three-dimensional information from these samples, it essentially unlocks a new dimension of data from material that has already been collected.
How CLARITY Compares to Other Clearing Methods
CLARITY is not the only tissue-clearing technique available, and the field has grown crowded. Organic solvent-based methods like iDISCO and uDISCO use chemicals that dissolve lipids and dehydrate tissue, often achieving transparency faster than hydrogel-based approaches. A systematic review comparing clearing methods found that these organic solvent-based approaches showed higher maximum effective imaging depths and lower levels of unwanted background fluorescence compared to hydrogel-based and hydrophilic methods. Imaging depth was also strongly linked to reduced background fluorescence across methods.
Another family of methods, called CUBIC, uses amino-alcohol solutions to clear tissue without hydrogel embedding. An optimized variant called CUBIC-f was shown to shorten both clearing and staining times while requiring less reagent than Advanced CLARITY. These aqueous methods are generally gentler on fluorescent proteins than organic solvents, which can quench fluorescence, but they may not achieve the same depth of transparency in thick specimens.
CLARITY’s main advantage over these alternatives is the hydrogel scaffold itself. Because proteins and nucleic acids are physically crosslinked into the gel, the tissue can withstand multiple rounds of staining, washing, and re-staining without degrading. You can label one set of targets, image the tissue, strip the antibodies, and stain for something completely different, all on the same sample. That capacity for repeated interrogation is harder to achieve with methods that do not embed the tissue in a supportive matrix. The trade-off is that CLARITY tends to be slower and more technically demanding than simpler clearing methods, and the hydrogel can introduce some tissue expansion or distortion that needs to be accounted for.
Expansion Microscopy and the Convergence of Techniques
An interesting parallel development is expansion microscopy, which also embeds tissue in a swellable polymer but deliberately expands the sample to achieve super-resolution imaging on ordinary microscopes. Rather than keeping the tissue its original size, expansion microscopy physically stretches it by a factor of four or more, spreading molecular targets apart enough that a conventional microscope can resolve structures that would normally require specialized super-resolution hardware. Researchers have extended this approach to work with conventional fluorescently labeled antibodies and fluorescent proteins, making it compatible with the same molecular tools used in CLARITY workflows.
Some labs have begun combining aspects of both approaches, using CLARITY-style clearing to make tissue transparent and then applying expansion to resolve fine details. The two methods occupy complementary niches: CLARITY excels at preserving large-scale architecture and connectivity across centimeters of tissue, while expansion microscopy excels at resolving nanoscale structures like synapses within smaller regions. Used together, they can span spatial scales from whole-brain circuitry down to individual synaptic contacts.
Lowering the Cost Barrier
One persistent criticism of CLARITY has been its cost and the specialized equipment it requires. The original electrophoresis setup, proprietary clearing reagents, and light-sheet microscopes represent a significant investment. Efforts to lower these barriers have come from multiple directions.
On the reagent side, the shift from FocusClear to TDE and other inexpensive matching solutions removed one of the biggest recurring costs. On the equipment side, researchers have used consumer-grade 3D printers to produce custom laboratory equipment adapted for CLARITY, including brain-slicing chambers and combined antibody-staining and imaging chambers, at a fraction of the cost of commercial alternatives. These 3D-printed parts can be iteratively designed and reproduced by any lab with access to a standard printer.
Perhaps the most ambitious cost-reduction effort combined simplified passive CLARITY with lens-free holographic on-chip microscopy, a low-cost imaging approach that replaces expensive microscope objectives with a simple sensor chip. The system used a common histological stain instead of fluorescent antibodies, enabling bright-field imaging of cleared tissue. The developers specifically positioned this approach as suitable for resource-limited settings, where the cost of a light-sheet microscope would be out of reach. Whether it matches the resolution and versatility of conventional CLARITY imaging is another question, but it demonstrates that the basic principle of tissue clearing can be adapted for very different budgets.
Tissue Clearing for Surgical Pathology
A more recent and somewhat surprising application of tissue-clearing principles is in the operating room. Traditional surgical pathology relies on frozen sections: a tissue biopsy is frozen, sliced thin, stained, and examined under a microscope, a process that takes at least 20 to 30 minutes. Researchers have explored whether tissue clearing could speed up or improve this workflow. One study developed a fast clearing protocol using benzyl alcohol and benzyl benzoate (BABB) with an orbital shaker that rendered clinical head and neck cancer biopsies transparent within a single hour. The clearing did not affect subsequent immunohistochemical staining, meaning the same biopsy could be optically imaged in three dimensions and then processed through standard pathology workflows. If this approach proves reliable at scale, it could eventually allow surgeons to get three-dimensional views of tumor margins while a patient is still on the operating table.