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 optimize 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 fluorescence species, from small molecules with low multiphoton cross sections to the brightest nanoparticles.

Fluorescent nucleobase analogs (FBAs) are established tools for studying oligonucleotide structure, dynamics and interactions, and have recently also emerged as an attractive option for labeling RNA-based therapeutics. A recognized drawback...

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