Time-, angle-, and energy-resolved two-photon photoelectron spectroscopy is used to study image-potential states of weakly bound monolayer graphene on the noble-metal surfaces Pt(111), Au/Ir(111), Au/Ni(111), and Ir(100). Binding energies are increased relative to the substrates, and lifetimes are similar for all systems under investigation. The properties of the image-potential states are mainly determined by the graphene layer with reduced influence of the substrate. Substrate-related occupied surface states close to the Fermi level are observed for graphene on fcc(111) substrates.

Ultrasensitive detection of DNA is achieved via two-photon excitation of a fluorescent base analogue.

Fluorescent nucleoside analogue ABN is readily detected at the single-molecule level and retains a quantum yield >50% in duplex DNA oligonucleotides.

The ability to routinely detect fluorescent analogs of nucleobases at the single-molecule level would create a wealth of opportunities to study nucleic acids. We report the multiphoton-induced fluorescence and single-molecule detection of a dimethylaminesubstituted extended-6-aza-uridine (DMA th aU). We show that DMA th aU can exist in a highly fluorescent form, emitting strongly in the visible region (470-560 nm). Using pulse-shaped broadband Ti:sapphire laser excitation, DMA th aU undergoes two-photon (2P) absorption at low excitation powers, switching to threephoton (3P) absorption at high incident intensity. The assignment of a 3P process is supported by cubic response calculations. Under both 2P and 3P excitation, the single-molecule brightness was over an order of magnitude higher than reported previously for any fluorescent base analog, which facilitated the first singlemolecule detection of an emissive nucleoside with multiphoton excitation.

Conformationally rigid unnatural α-amino acids bearing a pyrazoloquinazoline ring system that are amenable to both one- and two-photon excitation have been developed as new fluorescent probes.

The early detection of cancer cells and their visualization before and after surgery are essential for successful pre-and postoperative disease management. Although fluorescence imaging is a sensitive and versatile tool that is finding increasing use in clinical applications, there is a lack of tumour-targeting fluorophores. We report here a family of fluorescent Schiff base organotin dyes (1: Et 2 NÀ L-SnPh 2 , 2: Et 2 NÀ L-SnBu 2 , 3: MeOÀ L-SnPh 2 , 4: MeOÀ L-SnBu 2 , 5: HOÀ L-SnPh 2 , and 6: HOÀ L-SnBu 2 , where L = 2-hydroxybenzylidene-4-hydroxybenzhydrazine). In addition to one-photon-excited fluorescence, efficient twophoton excitation was demonstrated in compounds 1-4. Two of the compounds (5 and 6), both with hydroxyl substituents, were capable of selective accumulation in HeLa cells, allowing differentiation from normal cells (periodontal ligament cells). Compounds 1 and 3 showed excellent cancer cell staining (HeLa) using two-photon bioimaging, which is promising for biomedicine applications.

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.

No abstract