Good science hinges on the ability to observe beneath the surface of living organisms, ideally in intact specimens—to lay bare intrinsic biological information, structural morphology, organ and cell organization, all of which establishes the groundwork for tracking the effects of everything from genetic mutations to drug efficacy.
Attempts at observing and discerning the anatomy and physiology of animals dates back to as early as 500 BC in ancient Greece, where vivisections were common practice . Thankfully we’ve transitioned away from those early days, but still the objectives remain strikingly similar. Scientists struggle to generate images from the greatest depths of cells. Electron microscopy lets us travel further to see organelles at unimaginable resolution. But this and the majority of laboratory techniques focus on viewing slivers of the whole and analyzing smaller more manageable components. Brains, for example, are often sectioned at just a few microns thick before being stained or labeled and processed for microscopy, which has its own set of obstacles and the possibility of artifact introduction.
The clearing of anatomical samples was first reported over a century ago, but only recently has the intersection of advances in optical physics, chemical engineering, and microscopy technology produced methodology for broad applications in research . These developments in tissue clearing mean that labor intensive steps can be side-stepped while also producing more complete, more crystalline views of the biological landscape in organic models. In order to fully understand the connection between structure and function, interrogation of biological systems must be done in three-dimensions. Separating tissue may be altogether bypassed as whole organs, even entire animals, can be made as transparent as glass depending on the clearing protocol and tissue composition. Further, when sectioning of brains and other organs, or organelles, is required, it can be done in thicker intervals, preserving a greater amount of unperturbed biological data.
A closer look at optical clearing and considerations
Microscopy is limited by the scattering of light by tissue samples. In simple terms, the goal of tissue clearing is to allow light to pass through biological samples so that microscopes can see through to the nethermost layers. When light passes from one medium to another it will bend. The extent of this bending, or refraction, is dictated by the angle at which it enters the substance and the change in speed (for example, light travels more slowly in water than air). Tissue clearing works to minimize the difference, or the refractive index (RI), between all the different components of the tissue sample. Lipids, proteins, and intra- and extracellular fluids all have different RIs; as such tissue clearing works to remove the lipids and replace the fluids with a solution that has an RI equal to that of the remaining protein, about 1.5 .
Despite this straightforward principle, tissue clearing projects can become complicated during optimization. It must be established from the outset the types of molecules that are to be protected, such as mRNA or proteins, especially endogenously expressed fluorescent proteins. Scientists must also consider how much of the structural integrity and morphology will be preserved, and if immunolabeling will be possible after the clearing process—will antibodies be able to penetrate the sample and will the relevant epitopes be conserved? Each step in the clearing protocol—fixation, pretreatment, delipidation, labeling, and RI matching—must be thoroughly evaluated. Sometimes additional pretreatments may be necessary. For example, clearing animal limbs of entire animals requires decalcification of bone, which is a strong light scatterer. Large samples will require more customization, whereas small samples may only need an RI matching step .
Delipidation in solvents or detergents?
Scientists may submerge tissue in solvents, detergents, or a mixture, using a passive method or electrophoresis, which can speed up delipidation (but runs the danger of uneven removal). Each protocol has its own set of benefits and challenges.
Following fixation and permeabilization, solvent-based delipidation moves tissue through a series of solutions with increasing concentrations of a water-miscible solvent, such as ethanol, methanol, or tetrahydrofuran (THF), which may cause significant sample shrinkage. These reagents must also be carefully weighed. THF can cause morphological changes, while ethanol does not work well to clear tissue with a high lipid content. All of these reagents will quench fluorescence as they induce a conformational change in the fluorescent protein. Some of these effects can be mitigated through the use of carefully tailored protocols that pay attention to both pH and temperature. And although the above mentioned solvents work best for clearing large volumes of tissue, other solvents such as tert-butanol or propanol may be used to preserve fluorescence. The final step requires tissue incubation in the water-immiscible solvent, dichloromethane (DCM), which completes the lipid removal process .
Aqueous delipidation buffers contain detergents such as Triton-X-100 which work to disrupt the lipid bilayers of cells and generate micelles (spherical aggregates of lipid surrounded by the aqueous solution), which can then be transported out of the cell. The incubation step can take anywhere from hours to months, and depends on the kind of tissue, size of the organ or sample, volume of generated micelles (larger ones will take longer to diffuse out), and the extent of clearing needed. Delipidation, and optical transparency, may be boosted by using more alkaline pH or a higher temperature .
The final stage of the clearing process requires placing the tissue in a solution that matches the RI of the remaining cellular protein, about 1.50. These solutions are also divided into aqueous and solvent-based and there are challenges with both. Aqueous based methods struggle to reach a high enough RI but better preserve fluorescence. Solvents have a higher RI, but quench fluorescence. Advancements continue to improve the performance of all clearing methods, but optimization is always a critical step.
Simple immersion in high RI solutions
More recent developments have led to protocols in which organs or tissue may be cleared and RI matched using simple immersion in an aqueous buffer solution. The optical clearing agents (OCAs) often consist of glycerol and saccharides. As high concentration solutions are needed to achieve the desired RI of the tissue, the OCAs can be of a very viscous consistency and therefore quite difficult to work with in the lab. Some low-viscosity OCAs such as formamide however may reduce a fluorescence signal or cause tissue hardening . Some alternative commercial solutions are now available, however, which may circumvent some of these issues.
Binaree Rapid, for example, is an RI matching, low viscosity, aqueous OCA. With an RI of 1.52, no additional processing is needed prior to imaging. Compared to the commonly used detergent, Triton-X, the micelles produced by the gentle detergent in Binaree Rapid are significantly smaller and more permeable to tissue, able to penetrate through all layers, and diffuse out of the sample very quickly, cutting down on clearing time.
The solution can clear fixed brains in minimal time. Zebrafish brain is cleared in 1 hour, mouse brain in 6 hours, and rat brain in 48 hours. With the Binaree Rapid Clearing System, the entire post-fixation process, takes as few as 2 to 3 days, compared to up to several weeks for other methods, such as the detergent-based PACT or CUBIC protocols. The accompanying electrophoretic chamber, with automatic temperature control, and a simple user-friendly interface, further expedites delipidation of tissue. To use with a whole brain, simply transfer the fixed specimen to the chamber with the olfactory bulb facing the positively charged side. An enclosed cassette may be used for thin brain slices or other types of tissue, such as lung. Temperature controls, alongside the non-organic based OCA also protects important molecules, such as RNA, protein epitopes, and endogenous fluorescent chromophores, and is amenable to the use of dyes. Further, unlike other methods, there is minimal tissue swelling or shrinkage, and the specimen’s morphology remains impressively intact—in the brain, neuronal projections and dendrites are preserved.
Once the sample is cleared and mounted, images may be taken with a light sheet fluorescence microscope or confocal. And with the Binaree method, researchers can be confident that the data is reliable and reproducible.
Scientists can now look inside once opaque organs. New tissue clearing technology makes three-dimensional visualization of complex biological structures possible allowing for the elucidation of intrinsic, and pathological, structure-function relationships and the deciphering of complex tissue networks. Entire human brains  and even whole monkeys  can be rendered see-through advancing science in unimaginable ways. However, the process is not without myriad complications and necessary optimizations. Binaree Rapid makes generating optically transparent tissue, like whole animal brains, fast and simple, without confusing steps or hazardous chemicals. The future of science is here and it’s see-through; with Binaree it’s also fast and easy.
Morange, M, The Birth of Biology, 2021; https://press.princeton.edu/ideas/the-birth-of-biology
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