Comparative Analysis of 3D Models Now Possible

MIT neuroscientists and engineers have developed a new tool for clearing, labeling, 3D imaging, and analyzing organoids. Called "SCOUT" for "Single-Cell and Cytoarchitecture analysis of Organoids using Unbiased Techniques," the process can extract comparable features among whole organoids despite their uniqueness.

"When you are dealing with natural tissues you can always subdivide them using a standard tissue atlas, so it is easy to compare apples to apples," said Alexandre Albanese, co-lead author on a paper published in Scientific Reports. "But when every organoid is a snowflake and has its own unique combination of features, how do you know when the variability you observe is because of model itself rather than the biological question you are trying to answer? We were interested in cutting through the noise of the system to make quantitative comparisons."

The SCOUT process starts by making organoids optically transparent so they can be imaged with their 3D structure intact. The next SCOUT step is to infuse the cleared organoids with antibody labels targeting specific proteins to highlight cellular identity and activity. With organoids cleared and labeled, the team then images them with a light-sheet microscope to gather a full picture of the whole organoid at single-cell resolution.

The team chose its antibody labels strategically. With a goal of discerning cell patterns arising during organoid development, the team decided to label proteins specific to early neurons (TBR1) and radial glial progenitor cells (SOX2) because their organization impacts downstream development of the cortex. The team imbued SCOUT with algorithms to accurately identify every distinct cell within each organoid.

From there, SCOUT could start to recognize common architectural patterns such as identifying locations where similar cells cluster or areas of greater diversity, as well as how close or far different cell populations were from ventricles, or hollow spaces. In developing brains and organoids alike, cells organize around ventricles and then migrate out radially. With the aid of artificial intelligence-based methods, SCOUT was able to track patterns of different cell populations outward from each ventricle. Working with the system, the team therefore could identify similarities and differences in the cell configurations, or cytoarchitectures, across each organoid.

Ultimately the researchers were able to build a set of nearly 300 features on which organoids could be compared, ranging from the single-cell to whole-tissue level.