Issue 45, 2018

Pulse-shaped two-photon excitation of a fluorescent base analogue approaches single-molecule sensitivity

Abstract

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.

Graphical abstract: Pulse-shaped two-photon excitation of a fluorescent base analogue approaches single-molecule sensitivity

Supplementary files

Article information

Article type
Paper
Submitted
29 Aug 2018
Accepted
30 Oct 2018
First published
30 Oct 2018
This article is Open Access
Creative Commons BY license

Phys. Chem. Chem. Phys., 2018,20, 28487-28498

Pulse-shaped two-photon excitation of a fluorescent base analogue approaches single-molecule sensitivity

R. S. Fisher, D. Nobis, A. F. Füchtbauer, M. Bood, M. Grøtli, L. M. Wilhelmsson, A. C. Jones and S. W. Magennis, Phys. Chem. Chem. Phys., 2018, 20, 28487 DOI: 10.1039/C8CP05496G

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