Two-photon excitation of the fluorescent nucleobase analogues 2-AP and tC

Abstract

We present two-photon (2P) excitation measurements of two commonly-used and commercially available fluorescent nucleobase analogues in aqueous solution: the environment-sensitive adenine analogue 2-aminopurine (2-AP), which emits in the UV region, and the environment-insensitive cytosine analogue 1,3-diaza-2-oxophenothiazine (tC), which emits in the green region. We studied the 2P excitation of free 2-AP and found that the same emissive states are accessed as via one-photon (1P) and three-photon (3P) excitation. The 2P cross section of 2-AP was 0.2 GM, which is similar to that of related molecules such as NADH. We studied tC incorporated in three single-stranded DNA oligonucleotides by 1P and 2P spectroscopy and microscopy. We found that the same emissive states were populated via either the resonant or non-resonant pathway with a 2P cross section of 1.5 GM. Time-resolved fluorescence studies suggested that tC exists in solution as two species, which we assigned as the amino and imino tautomers. Using confocal microscopy, we found that similar emission rates could be achieved for 2P excitation, as compared with 1P excitation, but without the strong photobleaching observed in the latter case. Although we were unable to detect tC fluorescence at the single-molecule level, these results indicate that multiphoton excitation of nucleobase analogues is a promising approach for imaging applications.