Development of Diffusion Contrast Photo-Activated Localization Microscopy to Detect Sparse Protein Interactions at High Background

Many cellular signaling pathways contain protein binding partners that are expressed at extremely different concentrations. Interactions between these molecules can be difficult to observe using super-resolution microscopy techniques like Photo-Activated Localization Microscopy (PALM) since the overwhelming majority of localizations are from non-interacting molecules. Here, we develop and characterize Diffusion Contrast PALM (dcPALM) to overcome this barrier. By taking advantage of targeted photo-activation and the difference in diffusion coefficients between bound and unbound molecules, dcPALM reduces localizations from non-interacting molecules by 10-fold in yeast and even more in mammalian cells. dcPALM has little effect on the signal from interacting molecules and results in a dramatic increase in the PALM data quality of the interacting molecules. Using this technique, we were able to observe interactions involving fewer than 50 proteins per cell, even when over 95% of the molecules are not interacting. Our technique development and proof of concept experiments pave the way for future studies to quantify protein interactions in the signaling pathways that would not be able to be characterized by traditional PALM. Equal contributions by A.M. and J.M.R.