Coumarins fused with other aromatic units have recently emerged as a hot topic of research. Their synthesis is partly based on classical methodologies such as Pechmann reaction or Knoevenagel condensation, but it also sparked the discovery of completely new pathways. In very recent years so-called vertically-expanded coumarins were synthesized, effectively expanding the portfolio of existing architectures. A subtle relationship exists between the structure of fused coumarins and their optical properties. Although absorption of UV-radiation and light is unifying theme among these π-expanded coumarins, the fluorescence properties strongly depend on the structure. The mode of fusion, the type of additional ring and the presence of electron-donating and electron-withdrawing substituents all influence the photophysical parameters. Recent advances made it possible to modulate their absorption from 300 nm to 550 nm, resulting in new coumarins emitting orange light. This review serves as a guide through both synthesis strategies and structure-property relationship nuances. Strong intramolecular charge-transfer character made it possible to reach suitable values of two-photon absorption crosssection. Photophysical advantages of π-expanded coumarins have been already utilized in fluorescence probes and two-photon excited fluorescence microscopy.

Fluorescence imaging of tissues offer an essential means for studying biological systems. Autofluorescence becomes a serious issue in tissue imaging under excitation at UV-vis wavelengths where biological molecules compete with the fluorophore. To address this critical issue, a novel class of fluorophores that can be excited at ∼900 nm under two-photon excitation conditions and emits in the red wavelength region (≥600 nm) has been disclosed. The new π-extended dipolar dye system shows several advantageous features including minimal autofluorescence in tissue imaging and pronounced solvent-sensitive emission behavior, compared with a widely used two-photon absorbing dye, acedan. As an important application of the new dye system, one of the dyes was developed into a fluorescent probe for amyloid-β plaques, a key biomarker of Alzheimer's disease. The probe enabled in vivo imaging of amyloid-β plaques in a disease-model mouse, with negligible background signal. The new dye system has great potential for the development of other types of two-photon fluorescent probes and tags for imaging of tissues with minimal autofluorescence.