BPS2026 - Optimizing multimodal data collection on the ABEL trap for classification of DNA-fluorophore constructs

The scanned confocal geometry of the ABEL trap is well suited to multimodal data collection, but the scan geometry, timing, and detector configuration should be optimized for any given sample. Building upon our preexisting setup with capabilities for two color brightness, lifetime, and anisotropy, here we optimize scan geometry and timing for a range of samples undergoing energy transfer, including a series of DNA-fluorophore constructs called FRETfluors. We quantify the ability to classify these objects under different illumination conditions and detector configurations. Anisotropy measurements are carried out using a pair of APDs for each color, one for each polarization. The APDs also provide time-correlated single-photon counting (TCSPC) data for measuring the lifetime of samples. Using pulse interleaved excitation (PIE), we can separately interrogate the acceptor dye of a FRET pair, allowing for greater precision in measurements of FRET structures. We demonstrate that this system can be used to continuously monitor the positions and lifetimes of single molecules, including FRETfluor DNA nanostructures.