Pulse-shaped broadband multiphoton excitation for single-molecule fluorescence detection in the far field

Nobis, D., Sansom, H. G. and Magennis, S. W. (2023) Pulse-shaped broadband multiphoton excitation for single-molecule fluorescence detection in the far field. Methods and Applications in Fluorescence, 11(1), 017001. (doi: 10.1088/2050-6120/aca87f)

Abstract

Multiphoton excitation of fluorescence has many potential advantages over resonant (one-photon) excitation, but the method has not found widespread use for ultrasensitive applications. We recently described an approach to the multiphoton excitation of single molecules that uses a pulse shaper to compress and tailor pulses from an ultrafast broadband laser in order to optimise the brightness and signal-to-background ratio following non-linear excitation. Here we provide a detailed description of the setup and illustrate its use and potential by optimising two-photon fluorescence of a common fluorophore, rhodamine 110, at the single-molecule level. We also show that a DNA oligonucleotide labelled with a fluorescent nucleobase analogue, tC, can be detected using two-photon FCS, whereas one-photon excitation causes rapid photobleaching. The ability to improve the signal-to-background ratio and to reduce the incident power required to attain a given brightness can be applied to the multiphoton excitation of any fluorescent species, from small molecules with low multiphoton cross sections to the brightest nanoparticles.

• Item Type: Articles
• Additional Information: The authors gratefully acknowledge the support of the EPSRC for funding studentships (DN and HS).
• Keywords: Titanium sapphire laser, non-linear excitation, pulse shaping, ultrafast lasers.
• Status: Published
• Refereed: Yes
• Glasgow Author(s) Enlighten ID: Sansom, Mr Henry and Magennis, Dr Steven and Nobis, Mr David
• Authors: Nobis, D., Sansom, H. G., and Magennis, S. W.
• College/School: College of Science and Engineering > School of Chemistry
• Journal Name: Methods and Applications in Fluorescence
• Publisher: IOP Publishing
• ISSN: 2050-6120
• ISSN (Online): 2050-6120
• Published Online: 19 January 2023
• Copyright Holders: Copyright © 2023 The Authors
• First Published: First published in Methods and Applications in Fluorescence 11(1): 017001
• Publisher Policy: Reproduced under a Creative Commons License