Monday Article #68: Transparent mice and glowing internal systems

Whole-body clearing and scanning methods are crucial for the study of physiological systems in the maintenance of health and the pathogenesis of diseases. They allow researchers to investigate the delicate internal anatomical structures in the mice, without having to disturb or destroy them. Many breakthroughs have been achieved in establishing efficient techniques for creating these models, where four examples of these published breakthroughs will be discussed in this article.

Scale by Hama et al. (2011)

In 2011, Hama et al. developed a clearing reagent named Scale, which is reported to be particularly useful for light microscopy-based connectomics (study of connections in the nervous system) of cellular networks. Scale allowed the generation of transparent mouse brains and embryos, with fluorescent signals from labelled cells kept completely preserved. It was claimed that incubation of the brains and embryos in the Scale solution for >2 weeks could clear the tissues significantly, creating a soft and transparent product. The whole intact brain and its neuron population could be mapped in detail and with high resolution through this technique, where the effectiveness of this method was demonstrated by the researchers by mapping networks of the cortical, callosal, hippocampal, and neurogenic populations. Furthermore, the distance between blood vessels in the brain and neural stem cells could be quantified through this method, further enhancing the applicability of Scale.

Despite the numerous advantages of this clearing technique, two major drawbacks of the Scale method are the need for long time periods for complete clearance and the fragility of the finished product. Thus, further research was conducted with the aim of developing more advantageous methods.

PARS by Yang et al. (2014)

Figure 1. Image showing a whole-body cleared mouse, achieved using PARS (Yang et al., 2014).

In this PARS (perfusion-assisted agent release in situ) method, clarifying PACT (passive clarity technique) reagents are carefully pumped into the cranium or the blood vessels to achieve either whole-brain or whole-body clearing. The reagents can remove lipids (fats), that cause the tissues to block light, from the tissues, thus rendering them more transparent. Together with a refractive index matching solution (RIMS), the PARS method was able to turn opaque whole-mice into transparent and fluorescently-labelled ones for analysis at the cellular, subcellular, and single-molecule transcripts level. This method is much faster than that of Scale, where transparent tissues can be achieved by PARS within 2 to 3 days, and whole-mouse within 1 to 2 weeks.

uDISCO by Pan et al. (2016)

Figure 2. Visualisation of an intact mouse head after uDISCO clearing, with detailed structure of the optic nerve.

The ‘ultimate DISCO’ (uDISCO) method utilises a solvent with a refractive index of 1.579 for the clearing process. This method involves serial incubations of the samples into tert-butanol, an organic solvent, for dehydration, followed by another solution (DCM) for 45-60 minutes for removal of fats. The final step consists of immersion in a solution (BABB-D) for a minimum of 2 hours until transparency is achieved. The uDISCO technique was able to clear internal organs, tissues, and bones, as well as reveal and maintain fluorescence better than Scale. After clearing of the brain using uDISCO, morphologies of individual cells and blood vessels were also clearly visible. Due to a sample size reduction of up to 65% when using uDISCO, the maximum portion of cleared tissue that can be imaged at once is 2 to 4 times more than that of PARS, thus allowing whole-body imaging. Furthermore, due to dehydration, final cleared samples are rigid but still flexible enough for the adjustment of positions of longer samples, such as rat spinal cords.

vDISCO by Cai et al. (2018)

The vDISCO technique also involves immersion of the mice body in organic solvents for removal of lipids, while reducing the size by up to 60%. In this method, the mice body becomes hard with well preserved cell structure, where the body can be kept for years without compromising the viewing of individual cell morphologies. Instead of green-fluorescent proteins used by the previously discussed methods, Cai et al. utilised nanobodies found only in llamas, camels, and alpacas, which are about one-tenth of the size of a normal antibody molecule in many species. This allows specific labelling of desired types of cells, but with an added advantage of decreased size, allowing them to pass through small blood vessels and into organs easily. These nanobodies were pumped through the blood vessels of the mice, allowing imaging of subcellular details in transparent mice, as well as performing quantification. The entire body’s neuronal connectivity could be visualised, thus allowing holistic view of the body’s biological events.

wildDISCO by Mai et al. (2023)

WildDISCO is a whole-body antibody labelling method that utilises IgG antibodies, which are widely available on the market. To overcome the challenge of their larger size compared with nanobodies, a special tissue permeabilization technique was developed to allow uniform staining of the whole body with conventional IgG antibodies (around 150kDa in size). In addition, full homogeneous penetration of the antibodies could be achieved, travelling deeply into internal organs, which is more advantageous than the vDISCO technique that could not provide deep tissue staining with the standard IgG antibodies.

Figure 3. Whole-body cellular mapping in mouse using standard IgG antibodies (Mai et al., 2023).

Many interesting results were observed using this wildDISCO technique. For example, mice lacking microbes in their bodies had a less developed network of nerves in the gut as compared to normal mice, thus suggesting the role of the gut microbiome in the development of nerves. In addition, in cancer-bearing mice, immune cells could be seen gathering around the tumour cells.

Conclusion

Various methods, not limiting to the ones mentioned above, have been developed for clearing and labelling of mice organs and bodies. The visualisation of interactions and systems in intact mice bodies allows a greater understanding of the complex biological systems and their interplay in maintaining health. Furthermore, disease initiation and pathogenesis can also be studied in much higher detail to aid in the development of novel and improved therapeutics as well as diagnostic methods for various diseases.

References

Hama, H., Kurokawa, H., Kawano, H., Ando, R., Shimogori, T., Noda, H., Fukami, K., Sakaue-Sawano, A. and Miyawaki, A. (2011) ‘Scale: a chemical approach for fluorescence imaging and reconstruction of transparent mouse brain’, Nature neuroscience, 14(11), pp.1481-1488. [Online] DOI: 10.1038/nn.2928

Yang, B., Treweek, J.B., Kulkarni, R.P., Deverman, B.E., Chen, C.K., Lubeck, E., Shah, S., Cai, L. and Gradinaru, V. (2014) ‘Single-cell phenotyping within transparent intact tissue through whole-body clearing’, Cell, 158(4), pp.945-958. [Online] DOI: 10.1016/j.cell.2014.07.017

Pan, C., Cai, R., Quacquarelli, F.P., Ghasemigharagoz, A., Lourbopoulos, A., Matryba, P., Plesnila, N., Dichgans, M., Hellal, F. and Ertürk, A. (2016) ‘Shrinkage-mediated imaging of entire organs and organisms using uDISCO’, Nature methods, 13(10), pp.859-867. [Online] DOI: 10.1038/nmeth.3964

Cai, R., Pan, C., Ghasemigharagoz, A., Todorov, M.I., Förstera, B., Zhao, S., Bhatia, H.S., Mrowka, L., Theodorou, D., Rempfler, M. and Xavier, A. (2018) ‘Panoptic vDISCO imaging reveals neuronal connectivity, remote trauma effects and meningeal vessels in intact transparent mice’, BioRxiv, p.374785. [Online] DOI: 10.1101/374785

Mai, H., Luo, J., Hoeher, L., Al-Maskari, R., Horvath, I., Chen, Y., Kofler, F., Piraud, M., Paetzold, J.C., Modamio, J. and Todorov, M. (2023) ‘Whole-body cellular mapping in mouse using standard IgG antibodies’, Nature Biotechnology, pp.1-11. [Online] DOI: 10.1038/s41587-023-01846-0

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This article was written by Phoebe Tee