Two-photon excitation enables single-molecule detection of a fluorescent base analogue in DNA with high photostability

Fluorescent modification of nucleic acids using nucleobase analogues provides unique capabilities for imaging nucleic acids in cells and monitoring their conformational changes upon interaction with other biomolecules. The fluorescent nucleobase analogue ABN was shown recently to be the first base analogue to enable single-molecule detection of dsDNA, but its sensitivity to photobleaching under one-photon excitation required the use of oxygen scavenging reagents, and may limit future applications in live-cell imaging. Here, we show that two-photon excitation of ABN allows the stable detection of single DNA molecules in aqueous solution without antifade additives. The two-photon brightness of ABN in dsDNA exceeds by an order of magnitude that reported previously for any base analogue, and is comparable to that of the fluorescent protein EGFP, making it a promising candidate for the ultrasensitive imaging of nucleic acids in living cells and tissues.