title: Extending single molecule fluorescence observation time by amplitude-modulated excitation.
abstract: We present a hardware-based method that can improve single molecule fluorophore observation time by up to 1500% and super-localization by 47% for the experimental conditions used. The excitation was modulated using an acousto-optic modulator (AOM) synchronized to the data acquisition and inherent data conversion time of the detector. The observation time and precision in super-localization of four commonly used fluorophores were compared under modulated and traditional continuous excitation, including direct total internal reflectance excitation of Alexa 555 and Cy3, non-radiative Förster resonance energy transfer (FRET) excited Cy5, and direct epi-fluorescence wide field excitation of Rhodamine 6G. The proposed amplitude-modulated excitation does not perturb the chemical makeup of the system or sacrifice signal and is compatible with multiple types of fluorophores. Amplitude-modulated excitation has practical applications for any fluorescent study utilizing an instrumental setup with time-delayed detectors.
authors.forename: Lydia
authors.initials: L
authors.lastname: Kisley
authors.forename: Wei-Shun
authors.initials: WS
authors.lastname: Chang
authors.forename: David
authors.initials: D
authors.lastname: Cooper
authors.forename: Andrea P
authors.initials: AP
authors.lastname: Mansur
authors.forename: Christy F
authors.initials: CF
authors.lastname: Landes