Kisley, LydiaChang, Wei-ShunCooper, DavidMansur, Andrea P.Landes, Christy F.2015-07-102015-07-102013Kisley, Lydia, Chang, Wei-Shun, Cooper, David, et al.. "Extending single molecule fluorescence observation time by amplitude-modulated excitation." <i>Methods and Applications in Fluorescence,</i> 1, no. 3 (2013) IOP Publishing: 37001. http://dx.doi.org/10.1088/2050-6120/1/3/037001.https://hdl.handle.net/1911/80874We 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.engThis is an author's peer-reviewed final manuscript, as accepted by the publisher. The published article is copyrighted by IOP Publishing.Journal articlehttp://dx.doi.org/10.1088/2050-6120/1/3/037001