ETH Zurich creates high-resolution atlas of human retina tissue

17 May 2023

New protocol for flourescence imaging visualizes more than 50 proteins.

A project at ETH Zurich, the University of Basel and the University of Zurich has developed an imaging toolkit intended to assist high-resolution visualization of protein development.

The team applied its findings to the mapping of proteins in retina tissue, but the same principles could also be used to study how proteins develop in other tissues and organs.

Reported in Nature Biotechnology, the technique was developed for the imaging of organoids, simplified tissue systems generated from pluripotent stem cells as models of how proteins develop in the body.

Quantitative measurements of how organoid systems grow across different spatial scales are currently lacking, according to the project, and the new technique could offer a solution.

"The advantage of organoids is that we can intervene in their development and test active substances on them, which allows us to learn more about healthy tissue as well as diseases," said Barbara Treutlein from ETH Zurich.

The research builds on previous work at ETH Zurich studying iterative indirect immunofluorescence imaging, or 4i. This imaging protocol aims to refine the standard immunofluorescence imaging method used in biomedicine, and deliver more data from each sample examined by clinicians.

Although conventional immunofluorescence imaging usually marks three proteins, 4i was designed to use off-the-shelf antibodies and conventional fluorescence microscopes to visualize ten times more proteins by iterative hybridization and removal of antibodies from the sample, according to ETH Zurich data.

"Typically, researchers use fluorescence microscopy to highlight three proteins in a tissue, each with a different fluorescent dye, and it is not possible to stain more than five proteins at a time," noted the project team.

"In 4i technology, three dyes are used but these are washed from the tissue sample after measurements have been taken, and three new proteins are stained. This step was performed 18 times, by a robot, and the process took a total of 18 days. Lastly, a computer merges the individual images into a single microscopy image."

Improved understanding of retina development

In trials, the 4i approach was able to generate a protein map in which 53 different proteins were recorded at multiple stages of organoid development, at measurement scales from 150 nanometers to several millimeters, for a total of more than 400 million multiplexed measurements.

"We can use this time series to show how the organoid tissue slowly builds up, where different cell types proliferate and when, and where the synapses are located," said Gray Camp from the University of Basel. "The processes are comparable to those of retinal formation during embryonic development."

The project has made the data publicly available, intending its approach to be adopted by the research community for other tissue types, such as sections of the human brain and various tumor tissues. Ultimately the goal is an atlas of information about the development of human organoids and tissues.

It may also provide insights into the development of retinal diseases, if development of the retinal organoids is deliberately disrupted with drugs or genetic modifications and then imaged with the 4i technique.

"This will give us new insights into diseases such as retinitis pigmentosa, a hereditary condition that causes the retina’s light-sensitive receptors to gradually degenerate and ultimately leads to blindness," said Gray Camp.