Detail of a cross-section of a retinal organoid. Different tissue structures are made visible with different colours. Credit: Wahle et al. Nature Biotechnology 2023
Biological tissues – and their lab-cultivated organoid counterparts – may contain a range of different cell types and express dozens of different proteins, the spatial distribution of which can change throughout an organism’s development or in response to disease. Comprehensively mapping these structures throughout a single organoid or tissue sample can be difficult using conventional fluorescence microscopy techniques, as only a handful of proteins can typically be labeled at one time. Researchers at ETH Zurich, the University of Zurich and the University of Basel have used an advanced microscopy technique to generate a detailed atlas of a human retinal organoid, performing multiple rounds of staining on the same organoid sample to enable visualization of more than 50 proteins in one image.
The team utilized a technique known as iterative indirect immunofluorescence imaging, or 4i, which was developed a few years ago at the University of Zurich. The technique enables samples to be stained in multiple cycles, with three dyes used each cycle and then washed out of the sample before the next cycle. The histological section of retinal organoid was imaged using spinning disk confocal microscopy after each cycle; the process also included periodic elution cycles in which antibodies were not readded, in order to obtain a background signal and ensure the final images were corrected for background. The staining and elution cycles were automated using a liquid handling robot and a total of 18 staining cycles were completed in 18 days. At the end of the process, the images were combined into one final image showing all of the labeled proteins throughout the tissue. This method was used to image multiple organoids at different stages of development up to 39 weeks.
Using 4i, the researchers were able to visualize a total of 53 different proteins throughout the retinal organoid tissue. This visualization provides key information about the spatial distribution of cell types such as rods, cones and ganglion cells throughout the development of the retina. The team also supplemented the visual information with genetic information obtained through techniques such as single cell RNA sequencing (scRNA-seq) and single-cell assay for transposase-accessible chromatin using sequencing (scATAC-seq). The human retinal organoid atlas was published and is publicly accessible in an interactive format on the EyeSee4is website. The researchers noted that this study was the first use of the 4i technique to image human organoids. This research was published in Nature Biotechnology.
“We can use this time series to show how the organoid tissue slowly builds up, where which cell types proliferate and when, and where the synapses are located,” said senior author Gray Camp, a professor at the University of Basel. “The processes are comparable to those of retinal formation during embryonic development.”
The team hopes to use the data and techniques from this study to further examine the impact of drugs, genetic modifications and other disruptions in the development of retina organoids at the cellular level. They also believe the 4i technique can be used to comprehensively map other tissue types, such as brain tissue and tumors.