Monitoring Subcellular Stress Response via a Cell-permeant Rotor Dye.

Monitoring the response of cells to environmental challenges, e.g. after exposure to oxidative stress or pharmaceutical substances, not only provides clues for fundamental biological processes but can also serve as a valuable tool in drug development. Obtaining such insights on the subcellular level in a rapid and simple manner is therefore of major importance. Ideally, such an approach not only reports on compartment-specific responses but also allows for an inherent subcellular segmentation using the same data set. Here, we propose such a method based on fluorescence lifetimes of a single cell-permeant rotor dye with a broad emission spectrum. Using a k-means clustering approach, a straightforward, unsupervised, and rapid segmentation protocol allows for subcellular segmentation in addition to monitoring the differential response of these compartments to environmental stress, e.g. induced by hydrogen peroxide or the widely used chemotherapeutic cisplatin. Based on our data we suggest that our automatable approach can be a valuable and robust tool for pharmaceutical screening applications.