On the road for more efficient biocompatible two‐photon excitable fluorophores

AbstractRed‐to‐NIR absorption and emission wavelengths are key requirements for intravital bioimaging. One of the way to reach such excitation wavelengths is to use two‐photon excitation. Unfortunately, there is still a lack of two‐photon excitable fluorophores that are both efficient and biocompatible. Thus, we design a series of biocompatible quadrupolar dyes in order to study their ability to be used for live‐cell imaging, and in particular for two‐photon microscopy. Hence, we report the synthesis of 5 probes based on different donor cores (phenoxazine, acridane, phenazasiline and phenothiazine) and the study of their linear and non‐linear photophysical properties. TD‐DFT calculations were performed and were able to highlight the structure‐property relationship of this series. All these studies highlight the great potential of three of these biocompatible dyes for two‐photon microscopy, as they both exhibit high two‐photon cross‐sections (up to 3650 GM) and emit orange to red light. This potential was confirmed through live‐cell two‐photon microscopy experiments, leading to images with very high brightness and contrast.