Scientists at IMBA are passionate about discovery and advancing our understanding of biology. They are recognized leaders in their fields, regularly publishing in the top research journals. Contributions from IMBA research groups are of interest to everyone – including scientists, clinicians, and the public. The Research Highlights below summarize some of the most significant discoveries made by IMBA scientists.


20.02.2019

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Scientists from IMBA and the IMP developed a simple, rapid, non-toxic tissue clearing method which they tested in organoids, axolotls and fruit flies.

One of the biggest limitations when studying tissue architecture are tissues’ inherent properties of being turbid and opaque, which strongly limits the imaging depth - more than 100μm is barely possible.

For a long time, scientists had to rely on tissue sectioning, cutting the tissue in small sheets to look at its structure in detail, lacking the third dimension. Recent developments in tissue clearing now allow a close-up look at cellular organization –  such as neurons in the brain in their whole entity –  by rendering tissue transparent.

As biological samples are mostly composed of water, lipids and proteins, they contain a mix of components that have different refractive indices (RI). Through this effect, the deeper the light propagates into the tissue, the more it gets scattered, thus making it difficult to observe signals happening “inside” the sample.

Nowadays, there is a broad range of different tissue clearing techniques which usually follow the same strategy: Seeking transparency through homogenizing the RI of a sample by removing, replacing and modifying components with unwanted RI, such as fatty lipids or water. A problem: conventional tissue clearing techniques usually are very labor intense and time consuming, mostly toxic and often remove endogenous fluorophores.

Elly Tanaka’s lab –renowned for its research on tissue regeneration in the axolotl – and Jürgen Knoblich’s lab – famous for cerebral organoid technology –have been facing similar issues when it came to the analysis of large and complex 3D structures. The two labs joined forces and developed a novel, fast and simple procedure to clear a wide range of tissues in several species, including human cerebral organoids, Drosophila melanogaster, zebrafish, axolotl and Xenopus laevis.

The method, called 2Eci or 2nd generation ethyl cinnamate-based clearing, sets new standards as it can clear tissues in only 1 to 5 days. “Using this protocol, we could already look ad hundreds of cerebral organoids and their 3D architecture. Together with 3D immunohistochemistry, this technology allows me to look at different aspects of organoids in a- compared to previous technologies- high throughput manner and allows the observation of how neuronal stem cells become neurons and integrate into circuits.” says Daniel Reumann, PhD student in the Knoblich lab and co- author of the study. Another innovation of the 2Eci method is that it preserves a broad range of fluorescent proteins, that are key for fluorescence microscopy. “With this method, we can structure labels within different cells expressing different fluorophores, starting from axolotl muscles to dendritic spines in cerebral organoids, it is a faster and easily screenable way to look at the three-dimensional aspects of cell organization,” adds co-author Wouter Masselink, postdoctoral fellow in the Tanaka lab at IMP.

2Eci opens up tissue clearing to a much broader group of researchers due to its ease of use, the non-toxic nature of ethyl cinnamate and broad applicability.

 

Original publication: Masselink, W., Reumann, D., et.al. Broad applicability of a streamlined Ethyl Cinnamate-based clearing procedure, Development 2019
http://dev.biologists.org/content/early/2019/01/18/dev.166884