Multiphoton characterization and live cell imaging using fluorescent adenine analogue 2CNqA.

Fluorescent nucleobase analogues (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 of FBAs, however, is that they typically require excitation in the UV region, which for imaging in biological samples may have disadvantages related to phototoxicity, tissue penetration, and out-of-focus photobleaching. Multiphoton excitation has the potential to alleviate these issues and therefore, in this work, we characterize the multiphoton absorption properties and detectability of the highly fluorescent quadracyclic adenine analogue 2CNqA as a ribonucleotide monomer as well as incorporated, at one or two positions, into a 16mer antisense oligonucleotide (ASO). We found that 2CNqA has a two-photon absorption cross section that, among FBAs, is exceptionally high, with values of σ2PA(700 nm) = 5.8 GM, 6.8 GM, and 13 GM for the monomer, single-, and double-labelled oligonucleotide, respectively. Using fluorescence correlation spectroscopy, we show that the 2CNqA has a high 2P brightness as the monomer and when incorporated into the ASO, comparing favorably to other FBAs. We furthermore demonstrate the usefulness of the 2P imaging mode for improving detectability of 2CNqA-labelled ASOs in live cells.

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

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.

Synthesis of Thiazoloindole α-Amino Acids: Chromophores Amenable to One- and Two-Photon Induced Fluorescence.

Thiazoloindole α-amino acids have been synthesized in four steps from tryptophan using a dual-catalytic thiolation reaction and a copper-mediated intramolecular N-arylation process. Late-stage diversification of the thiazoloindole core with electron-deficient aryl substituents produced chromophores that on one-photon excitation displayed blue-green emission, mega-Stokes shifts, and high quantum yields. The thiazoloindole amino acids could also be excited via two-photon absorption in the near-infrared, demonstrating their potential for biomedical imaging applications.