Tissue clearing is a process that makes tissue samples transparent by removing opaque molecules. This technique minimizes light scattering and light absorption to enable better light penetration during microscopy¹. The resulting minimization of light absorption in the sample allows the generation of 3D microscopy images that depict biological structures more accurately compared to conventional 2D methods. Advanced 3D microscopy, in combination with rapidly advancing tissue-clearing techniques, facilitates high-resolution optical exploration of intact tissues, organs, and even whole organisms, illuminating subcellular details in whole organs and even small mammals.¹

NeuN antibody (ab177487) (green) and DAPI (blue) used with our Tissue Clearing Kit (ab243298) to clear and stain a 1 mm thick coronal section of mouse brain.

Further insights can be gained by immunolabeling various targets in cleared tissue samples. This enables visualization of sub-cellular structures and proteins while preserving their spatial distribution within the specimen. This provides an improved representation of the in vivo microenvironment of the specimen, allowing for better prediction and research of targets of interest^2,3,4,5.

Why clear 3D cell cultures

3D cell culture models (eg organoids, microtissues, spheroids) give a much better representation of the in vivo microenvironment and thus offer improved predictive capability compared to traditional 2D cell culture models. However, one of the problems with 3D cell culture models has been their characterization. Assays that rely on the dissolution of the cells in a 3D structure lose the spatial features that make these models so valuable.

Despite 3D cultures being relatively thin (compared to a whole organ), light still attenuates after 1–3 cell layers and thus most imaging techniques (widefield, confocal) characterize only the cells on the periphery of these models. Since peripheral cells receive the highest levels of compounds and nutrients, they do not represent the whole model. Peripheral cells often exhibit atypical responses to compounds than the average cell within the model. While optical sectioning solves the issue of light attenuation and scattering, this only provides information about single planes of a 3D model.

With tissue clearing methods, it's possible to imagine the entire 3D cell culture and simultaneously preserve structural integrity.

Albumin antibody (ab207327) (yellow), CD68 antibody (green), Vimentin antibody (red) and DAPI (blue) used with  3D Cell Culture Clearing Kit (ab243299)to clear and stain a 3D hepatocyte cell culture spheroid.

Tissue clearing methods and tips

The clearing of tissues and even whole organs is achieved through several chemical and (in some cases) electrophoretic steps. Tissue clearing is conducted by incubating the specimen in a series of specialized solutions to eliminate interfering molecules such as lipids, pigments, and calcium phosphate. There are three prevalent approaches to tissue clearing: hydrophobic techniques employing organic solvents, hydrophilic techniques using aqueous solutions, and hydrogel-based methods. Each approach has its own advantages and limitations. For an in-depth look at these, you may find it useful to browse the protocols and FAQs of the main types (Table 1).

References

1. Ueda, H. R.,, Ertürk, A.,, Chung, K.,, et al. Tissue clearing and its applications in neuroscience Nature Reviews Neuroscience  21 ,61-79 (2020)
2. Susaki, E. A.,, Tainaka, K.,, Perrin, D.,, et al. Whole-brain imaging with single-cell resolution using chemical cocktails and computational analysis. Cell  157 ,726-739 (2014)
3. Belle, M.,, Godefroy, D.,, Dominici, C.,, et al. A simple method for 3D analysis of immunolabeled axonal tracts in a transparent nervous system. Cell Reports  9 (4),1191-201 (2014)
4. Renier, N.,, Wu, Z.,, Simon, D. J.,, et al. iDISCO: a simple, rapid method to immunolabel large tissue samples for volume imaging. Cell  159 (4),896-910 (2014)
5. Chung, K.,, Wallace, J.,, Kim, S-Y.,, et al. Structural and molecular integration of intact biological systems. Nature  497 ,332-337 (2013)