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Updated: Jan 21, 2026

Single Molecule Fluorescence Microscopy on Planar Supported Bilayers
Published on: October 31, 2015
Single-Molecule Detection of a Fluorescent Nucleobase Analogue via Multiphoton Excitation
David Nobis1, Rachel S Fisher2, Mats Simmermacher2
1WestCHEM School of Chemistry, University of Glasgow, Joseph Black Building, University Avenue, Glasgow G12 8QQ, U.K.
Abstract:
The ability to routinely detect fluorescent nucleobase analogues at the single-molecule level would create a wealth of opportunities to study nucleic acids. We report the multiphoton-induced fluorescence and single-molecule detection of a dimethylamine-substituted extended-6-aza-uridine (DMAaU). We show that DMAaU can exist in a highly fluorescent form, emitting strongly in the visible region (470-560 nm). Using pulse-shaped broadband Ti:sapphire laser excitation, DMAaU undergoes two-photon (2P) absorption at low excitation powers, switching to three-photon (3P) absorption at high incident intensity. The assignment of a 3P process is supported by cubic response calculations. Under both 2P and 3P excitation, the single-molecule brightness was over an order of magnitude higher than reported previously for any fluorescent base analogue, which facilitated the first single-molecule detection of an emissive nucleoside with multiphoton excitation.
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