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

Fisher, R. S., Nobis, D., Füchtbauer, A. F., Bood, M., Grøtli, M., Wilhelmsson, L. M., Jones, A. C. and Magennis, S. W. (2018) Pulse-shaped two-photon excitation of a fluorescent base analogue approaches single-molecule sensitivity. Physical Chemistry Chemical Physics, 20(45), pp. 28487-28498. (doi: 10.1039/c8cp05496g) (PMID:30412214)

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.

• Item Type: Articles
• Additional Information: This work was supported by EPSRC (studentships for RSF and DN) and the University of Edinburgh (RSF). This work was also supported by the Swedish Foundation for Strategic Research (SSF, grants no. ID14-0036 (MG), IS14-0041 (LMW) and IRC15-0065 (LMW)) and the Swedish Research Council (VR, grant No. 2017-03707 (LMW)). We would like to thank Dr Dmitry Pestov (Biophotonic Solutions Inc.) for technical support. Research data supporting this publication can be found at http://dx.doi.org/10.7488/ds/2457.
• Status: Published
• Refereed: Yes
• Glasgow Author(s) Enlighten ID: Magennis, Dr Steven and Nobis, Mr David and Fisher, Rachel
• Authors: Fisher, R. S., Nobis, D., Füchtbauer, A. F., Bood, M., Grøtli, M., Wilhelmsson, L. M., Jones, A. C., and Magennis, S. W.
• College/School: College of Science and Engineering > School of Chemistry
• Journal Name: Physical Chemistry Chemical Physics
• Publisher: Royal Society of Chemistry
• ISSN: 1463-9076
• ISSN (Online): 1463-9084
• Copyright Holders: Copyright © 2018 The Owner Societies
• First Published: First published in Physical Chemistry Chemical Physics 20:28487-28498
• Publisher Policy: Reproduced under a Creative Commons License
• Data DOI: 10.7488/ds/2457