Methods for automating the analysis of live-cell single-molecule FRET data

Single-molecule FRET (smFRET) is a powerful imaging platform capable of revealing dynamic changes in the conformation and proximity of biological molecules. The expansion of smFRET imaging into living cells creates both numerous new research opportunities and new challenges. Automating dataset curation processes is critical to providing consistent, repeatable analysis in an efficient manner, freeing experimentalists to advance the technical boundaries and throughput of what is possible in imaging living cells. Here, we devise an automated solution to the problem of multiple particles entering a region of interest, an otherwise labor-intensive and subjective process that had been performed manually in our previous work. The resolution of these two issues increases the quantity of FRET data and improves the accuracy with which FRET distributions are generated, increasing knowledge about the biological functions of the molecules under study. Our automated approach is straightforward, interpretable, and requires only localization and intensity values for donor and acceptor channel signals, which we compute through our previously published smCellFRET pipeline. The development of our automated approach is informed by the insights of expert experimentalists with extensive experience inspecting smFRET trajectories (displacement and intensity traces) from live cells. We test our automated approach against our recently published research on the metabotropic glutamate receptor 2 (mGluR2) and reveal substantial similarities, as well as potential shortcomings in the manual curation process that are addressable using the algorithms we developed here.