Single-molecule detection provides researchers with a unique method to probe kinetics of biomolecules in their native environment, without the need to synchronize the molecular states. In contrast to other confocal-feedback 3D single-particle tracking demonstrations, we tracked single DNA reporter strands inside a live cell and measured their annealing-melting kinetics. Our 3D-SMT method uses multiple single-photon detectors or multiplexed pulsed laser illuminations to achieve spatial filtering, which not only allows for high-resolution 3D localization of single molecules in live cells, but enables simultaneous characterization of molecular binding state through a continuous lifetime measurement. The data acquired can be used to generate new models that can predict in-cellulo hybridization kinetics from sequence, study the molecular crowding inside cells and probe the cellular development and transition states. More details can be found in Chen et al, Nanoscale, 2023.
