University of Zurich (UZH) scientists report that they have developed a new technique for analyzing cells and their components called Iterative Indirect Immunofluorescence Imaging (4i). They say this innovation greatly refines the standard immunofluorescence imaging method used in biomedicine and provides clinicians with a large amount of data from each individual sample. 4i makes it possible to observe the spatial distribution of at least 40 proteins and their modifications in the same cell for hundreds of thousands of cells simultaneously at various levels, from the tissue down to the organelle level, the team adds.

Their study (“Multiplexed protein maps link subcellular organization to cellular states”) is published in Science.

“We report a simple, robust, and nondegrading protocol that achieves 40-plex protein staining in the same biological sample using off-the-shelf antibodies called iterative indirect immunofluorescence imaging (4i). In conjunction with high-throughput automated microscopy and computer vision, 4i allows highly reproducible multiplexed measurements from surface areas of several mm2 subsampled by pixels of 165 nm by 165 nm,” write the investigators.

“This approach simultaneously captures functionally relevant properties that emerge at the cell population, cellular, and intracellular level. 4i can thus quantify the influence of local cell crowding on protein abundance, the effect of cell cycle position on protein phosphorylation, patterns of protein subcompartmentalization, and organelle morphology all in the same single cell and across thousands of cells.”

“4i is the first imaging technique which gives us a multiplexed tissue-to-organelle view of biological samples. We can, for the first time, link multiplexed information derived at the tissue, cellular, and subcellular level in one and the same experiment,” says Gabriele Gut, PhD., lead author of the study and postdoctoral researcher at the Institute of Molecular Life Sciences at UZH. 

Immunofluorescence (IF) uses antibodies to visualize and locate proteins in biological samples. While the standard IF method usually marks three proteins, 4i uses off-the-shelf antibodies and conventional fluorescence microscopes to visualize ten times more proteins by iterative hybridization and removal of antibodies from the sample. “Imagine cell biologists to be journalists. Every experiment is an interview with our cells. With conventional IF I can ask three questions, whereas with 4i I can have a discussion on more than 40 topics,” explains Dr. Gut.

Once acquired, the huge amount of data must also be able to be analyzed. “We generated images with subcellular resolution for thousands of cells for 40 channels for more than 10 treatment conditions,” he continues. “The human eye and brain cannot process the biological complexity collected by 4i”. 

To make full use of the 4i data, Dr. Gut developed a new computer program for visualization and analysis called Multiplexed Protein Maps. It extracts the multiplexed fluorescence signal for millions of pixels and generates an abstract but representative map of the multiplexed protein distribution in cells. 

The researchers were thus able to generate a systematic survey of the cellular landscape. They managed to visualize the spatial intracellular organization of most mammalian organelles along the cell cycle and in different microenvironments. 

The applications for 4i and multiplexed protein maps range from basic research to precision medicine. “We hope that 4i and multiplexed protein maps will help researchers to understand processes better that have been at the center of biological research for decades,” says Dr. Gut. 

At the same time, the researchers plan to use these technologies to advance the frontiers of precision medicine, particularly in cancer diagnosis and therapy selection.

The 4i analysis method can also be used to determine the effects of pharmacological substances on the organization and physiology of cells, according to Lucas Pelkmans, Ph.D., professor at the Institute of Molecular Life Sciences at UZH. It is currently being used in a translational research collaboration with clinicians and a pharmaceutical company with the aim of improving treatment outcome of cancer patients. 

Dr. Pelkmans and his research team aim to characterize tumor cells of patients who have been treated with different cancer medications. The scientists hope that the lab results will provide information to support clinical decision-making for the individual treatment of patients. Moreover, the researchers plan to implement 4i and multiplexed protein maps on tissue sections of tumors to identify relevant biomarkers and thus improve diagnoses and prognoses for cancer patients.

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