Working with X-CLARITY – Key Considerations for Tissue Clearing

I can see clearly now the fat has gone…

Sounds like the lyrics to a popular song, but it is a call to understand just what tissue clearing is and to negotiate the minefield of promises and misinformation around this topic. 

Many articles explain interactions of light, refractive indices, scattering, constructive and destructive waves, and describing the core physics behind the observed phenomena. One such paper also discusses the history of tissue clearing until the very early days of the CLARITY technique developed by the Deisseroth lab at Stanford University. CLARITY is a method used to resolve issues encountered when clearing tissues of fat using solvents.

Fundamentally, to see deep into intact tissue for microscopy capable of imaging beyond 1mm, say 5mm, required a new technique—a method that could avoid the inherent shrinkage and short fluorescent lifetimes of a solvent-based approach. 

Opaqueness results from the multiple scattering of light; when a wave interacts a particle and propagates light in all directions, the tissue (a collection of compacted cells), considers this a multitude of light sources. Some methods utilise long wavelengths in an attempt to avoid this type of short wavelength scattering.

How does this advantage work?  

Try to test scattering by shining a torch through your hand in a darkened room. Your hand will appear bright red. Opacity is very apparent in bone and teeth, densely packed cells carrying calcium that scatters light. It is virtually impossible to shine a light through these tight areas. Dense samples must undergo decalcification before performing clearing steps. 

Solvent-based techniques have an ever-increasing list of novel solvents all attempting to make a silk purse from a sow’s ear. All are fundamentally flawed as the restrictive selection of antibodies and short fluorescent lifetimes is limiting. 

The solvents rarely are microscope-friendly, and if they are, you have invested heavily into objectives that claim to be solvent-resistant. These solvents cannot clear beyond around 1mm. These shortcomings don’t seem to deter the vigour of their desire to sell these products. If you wish to image through a whole mouse brain, for example, the most effective method is X-CLARITY, which can clear through the tissue in just six hours! Plus, it is aqueous, allowing a vast array of antibodies. Larger tissue sections are desirable to understand interactions across a complete organ, or at times the whole organism while avoiding the losses associated with sectioning.

Antibodies are used for molecular specificity, to bind to a specific protein, effectively singling it out for fantastic fluorescent images. They pop out from the background illumination.  

DeepLabel™ Antibody Staining Kit

Whether the clearing method is solvent or aqueous based, many techniques affect the ability for a molecular probe to infiltrate the tissue. Often, this is a laboriously slow diffusion of the probe into a thin slither of tissue. Staining thick tissues is notoriously fraught with inconsistencies, lack of uniformity from non-specific binding to the outer edges.

The DeepLabel™ Antibody Staining Kit is a collection of non-toxic reagents optimised to enable macromolecular probes to penetrate the thick tissue. An analogy is these reagents create the highway for the labels to travel on, as they  search for specific binding sites. The added benefit is the Deeplabel Antibody Staining kit makes this process faster, more efficient by using far less antibodies. The secret to such vibrant fluorescent images is the 2.6 X greater signal to background ratio. Thanks to homogenous staining that permits subcellular resolution, DeepLabel™ is compatible with all antibodies and cleared tissues.

Refractive Index Matching

Once labelled, the tissue is placed in a Refractive Index Matching Media (RIMM) to enhance the clarity for microscopy, making some tissues almost vanish. In the context of a microscope, this process eliminates the scattering of illumination and accomplishes the mission – where the fluorescence ‘pops’ out to produce vibrant, crisp images. Logos Biosystems supply this ~1.46 RI media which enhances confocal imaging of tissues, allowing for reduced laser power, low photobleaching, and preservation of fluorescent signals for up to 2 weeks.

While clearing methods allow light to penetrate samples for imaging, the method used can affect tissue permeability to molecular probes. Conventional labelling protocols involve the slow diffusion of probes into thin sample sections, which translates to a time-consuming impracticality when applied to thicker samples. The slow progression of diffusion can also lead to uneven staining, with higher, nonspecific binding on the outside of a thicker sample. 

The X-CLARITY system 

The X-CLARITY™ systems and reagents standardize, simplify, and accelerate each step of the tissue clearing process. With the CLARITY method, preserved tissues are embedded in a hydrogel matrix and lipids are actively extracted through electrophoresis to create a stable and optically transparent tissue-hydrogel hybrid that is chemically accessible for multiple rounds of antibody labelling and imaging. Native cytoarchitecture remains intact and even endogenous fluorescence proteins are preserved for robust fluorescence imaging downstream.

X-CLARITY system has three main components

1. A polymeriser activates the temperature- triggered hydrogel, which forms the structure of the tissue after fat removal. The result is a material similar to a western blot gel. It is harmless in this form and maintains shape during the electrophoresis.
2. The ETC Chamber is where the Electrophoretic Tissue Clearing takes place. Placing the sample between two large platinum electrode plates maximises the surface area and ensures even clearings while pumping an aqueous detergent through the chamber. 
3. The Control Tower contains pumps and temperature regulators. As detergent is pumped through the chamber, a temperature probe provides feedback. Cooling the tissue achieves the setpoint. It is essential to maintain control over temperature and ensure it does not exceed 37°C to protect the protein.

Logos Biosystems have made clearing a turn-key operation of four steps: polymerise, clear, label, and RIMM. Applications for the X-CLARITY method extend beyond brains to clear virtually any organ, even organoids. If you need assistance in determining if your application is possible, contact us.
The Logos Biosystems X-CLARITY system’s unique design accelerates the removal of lipids from tissues in a highly efficient manner. X-CLARITY is an all-in-one system with ready-to-use reagents for simple, rapid, and efficient tissue clearing. To learn more about the X-CLARITY system, speak to ATA Scientific today.