Subdiffraction fluorescence lifetime imaging with Flipper-TR

The combination of stimulated emission depletion (STED) microscopy with fluorescence lifetime imaging microscopy (FLIM) offers a powerful route to functional super-resolution imaging, but remains technically challenging for lifetime-based sensing because of the complex interplay between stimulated emission, photon budget, and accurate lifetime quantification. In this technical note, we evaluate and optimize STED-FLIM for nanoscale mapping of bacterial membrane mechanics using the mechanosensitive probe Flipper-TR. This probe is particularly demanding for STED-FLIM because it exhibits a biexponential fluorescence decay, with only the long lifetime reporting on membrane mechanical properties. Using liveBacillus subtiliscells, we establish an acquisition and analysis workflow that combines optimized imaging conditions with tail fitting to minimize distortions introduced by the depletion pulse. Under these conditions, STED imaging achieved a more than twofold improvement in spatial resolution compared with confocal microscopy, while maintaining sufficient photon counts for robust lifetime estimation. Finally, we discuss the trade-offs between spatial resolution and photon budget and provide practical recommendations for implementing STED-FLIM with environmentally sensitive probes. These results extend the application of Flipper-TR to functional super-resolution microscopy and provide a framework for robust STED-FLIM measurements of membrane nanomechanics and other fluorescence lifetime-based sensing applications.