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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.

SUBMITTER: Kisley L

PROVIDER: S-EPMC3935333 | biostudies-literature | 2013 Sep

REPOSITORIES: biostudies-literature

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Extending single molecule fluorescence observation time by amplitude-modulated excitation.

Kisley Lydia L Chang Wei-Shun WS Cooper David D Mansur Andrea P AP Landes Christy F CF

Methods and applications in fluorescence 20130901 3

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, inc ...[more]

PMID: 24587894

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Project description:BACKGROUND:Single-molecule detection (SMD) technologies are well suited for clinical diagnostic applications by offering the prospect of minimizing precious patient sample requirements while maximizing clinical information content. Not yet available, however, is a universal SMD-based platform technology that permits multiplexed detection of both nucleic acid and protein targets and that is suitable for automation and integration into the clinical laboratory work flow. METHODS:We have used a sensitive, specific, quantitative, and cost-effective homogeneous SMD method that has high single-well multiplexing potential and uses alternating-laser excitation (ALEX) fluorescence-aided molecule sorting extended to 4 colors (4c-ALEX). Recognition molecules are tagged with different-color fluorescence dyes, and coincident confocal detection of ?2 colors constitutes a positive target-detection event. The virtual exclusion of the majority of sources of background noise eliminates washing steps. Sorting molecules with multidimensional probe stoichiometries (S) and single-molecule fluorescence resonance energy transfer efficiencies (E) allows differentiation of numerous targets simultaneously. RESULTS:We show detection, differentiation, and quantification-in a single well-of (a) 25 different fluorescently labeled DNAs; (b) 8 bacterial genetic markers, including 3 antibiotic drug-resistance determinants found in 11 septicemia-causing Staphylococcus and Enterococcus strains; and (c) 6 tumor markers present in blood. CONCLUSIONS:The results demonstrate assay utility for clinical molecular diagnostic applications by means of multiplexed detection of nucleic acids and proteins and suggest potential uses for early diagnosis of cancer and infectious and other diseases, as well as for personalized medicine. Future integration of additional technology components to minimize preanalytical sample manipulation while maximizing throughput should allow development of a user-friendly ("sample in, answer out") point-of-care platform for next-generation medical diagnostic tests that offer considerable savings in costs and patient sample.

Dataset Information

Extending single molecule fluorescence observation time by amplitude-modulated excitation.

Publications

Extending single molecule fluorescence observation time by amplitude-modulated excitation.

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