The combination of two two-photon-induced processes in a FRET-operated photochromic fluorene-dithienylethene dyad lays the fundament for the observation of a quartic dependence of the fluorescence signal on the excitation light intensity. While this photophysical behavior is predicted for a four-photon-absorbing dye, the herein proposed approach opens the way to use two-photon absorbing dyes, reaching the same performance. Hence, the spatial resolution limit, being a critical parameter for applications in fluorescence imaging or data storage with common two-photon-absorbing dyes, is dramatically improved.

A water soluble diarylethene (DAE) derivative that displays exceptionally intense fluorescence from the colorless open form has been synthesized and characterized using UV/vis spectroscopy and fluorescence microscopy. We show that the bright emission from the open form can be rapidly switched using amplitude modulated red light, that is, by light at wavelengths longer than those absorbed by the fluorescent species. This is highly appealing in any context where undesired background fluorescence disturbs the measurement, e.g., the autofluorescence commonly observed in fluorescence microscopy. We show that this scheme is conveniently applicable using lock-in detection, and that robust amplitude modulation of the probe fluorescence is indeed possible also in cell studies using fluorescence microscopy.

Here, we present the non-linear spectroscopic characterization of the fluorescent nucleobase analogue 2CNqA as a monomer and inside an antisense oligonucleotide. Its applicability as a 2P fluorescent reporter is demonstrated using live cell imaging.

Azido-functionalised push–pull fluorenes, modified via CuAAC, show triazole-induced emission shifts and enhanced photophysical properties. These findings enable the synthesis of high-performance fluorosolvatochromic dyes for diverse applications.