The Anti-Brownian ELectrokinetic (ABEL) trap uses electrophoresis or electroosmosis to trap fluorescent single molecules in free solution. Several photophysical parameters, such as brightness, lifetime, anisotropy, emission spectrum, can be simultaneously measured with very high precision. Here we demonstrate the added capabilities of the ABEL trap upon modifying the excitation technique. First, we utilize dynamic excitation brightness to observe photophysically induced responses in a trapped light-harvesting protein-pigment complex from cyanobacteria. Second, we trap fluorescently labeled biomolecules using two colors via pulsed interleaved excitation (PIE) to enable acceptor-corrected FRET measurements. These advances in excitation patterning for the ABEL trap allow for novel sensing abilities that combine the benefits of cutting-edge single-molecule imaging and spectroscopy with the long isotropic view of single molecules provided by the ABEL trap.
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