Ultrasensitive detection of a responsive fluorescent thymidine analogue in DNA

Fluorescent base analogues (FBAs) are versatile nucleic acid labels that can replace a native nucleobase, while maintaining base pairing and secondary structure. Following the recent demonstration that free FBAs can be detected at the single-molecule level, the next goal is to achieve this level of detection sensitivity in oligonucleotides. Due to the short-wavelength absorption of most FBAs, multiphoton microscopy has emerged as a promising approach to single-molecule detection. We report the multiphoton-induced fluorescence of 5-(5-(4-methoxyphenyl)thiophen-2-yl)-6-aza-uridine (MeO