https://neurosciencenews.com/organoid-retina-microscopy-23194/
--RikMany proteins visible simultaneously
At the heart of the methods the scientists used for their approach was the 4i technology: iterative indirect immunofluorescence imaging. This new imaging technique can visualize several dozen proteins in a thin tissue section at high resolution using fluorescence microscopy.
The 4i technology was developed a few years ago by Lucas Pelkmans, a professor at the University of Zurich and coauthor of the study that has just been published in the scientific journal Nature Biotechnology.
It is in this study that the researchers applied this method to organoids for the first time.
Typically, researchers use fluorescence microscopy to highlight three proteins in a tissue, each with a different fluorescent dye.
For technical reasons, it is not possible to stain more than five proteins at a time. 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 on which 53 different proteins are visible.