Pulse-shaped two-photon excitation of a fluorescent base analogue approaches single-molecule sensitivity
Journal article, 2018

Fluorescent nucleobase analogues (FBAs) have many desirable features in comparison to extrinsic fluorescent labels, but they are yet to find application in ultrasensitive detection. Many of the disadvantages of FBAs arise from their short excitation wavelengths (often in the ultraviolet), making two-photon excitation a potentially attractive approach. Pentacyclic adenine (pA) is a recently developed FBA that has an exceptionally high two-photon brightness. We have studied the two-photon-excited fluorescence properties of pA and how they are affected by incorporation in DNA. We find that pA is more photostable under two-photon excitation than via resonant absorption. When incorporated in an oligonucleotide, pA has a high two-photon cross section and emission quantum yield, varying with sequence context, resulting in the highest reported brightness for such a probe. The use of a two-photon microscope with ultrafast excitation and pulse shaping has allowed the detection of pA-containing oligonucleotides in solution with a limit of detection of ∼5 molecules, demonstrating that practical single-molecule detection of FBAs is now within reach.

Author

Rachel S. Fisher

University of Edinburgh

David Nobis

University of Glasgow

Anders Foller Füchtbauer

Chalmers, Chemistry and Chemical Engineering, Chemistry and Biochemistry, Physical Chemistry

Mattias Bood

University of Gothenburg

Morten Grötli

University of Gothenburg

Marcus Wilhelmsson

Chalmers, Chemistry and Chemical Engineering, Chemistry and Biochemistry

Anita C. Jones

University of Edinburgh

Steven W. Magennis

University of Glasgow

Physical Chemistry Chemical Physics

1463-9076 (ISSN) 1463-9084 (eISSN)

Vol. 20 45 28487-28498

Subject Categories

Atom and Molecular Physics and Optics

Other Physics Topics

Theoretical Chemistry

DOI

10.1039/c8cp05496g

PubMed

30412214

More information

Latest update

12/10/2018