Fluorescence bioimaging combined with fluorescence microscopy technology has revolutionized human ability to study and visualize complex life phenomena at the molecular level to understand the cellular events with least ambiguity. The continuous synergistic growth of bioscience and medical science demands for better diagnostic tools/systems; resulting the introduction of two-photon fluorescence bioimaging recently based on two-photon induced excitation technology. Two-photon bioimaging is highly advantageous to provide 3D imaging of living specimens (including animal tissues) with sub-micrometer resolution down to the depth of a millimeter with high-sensitivity and reduced photo-damage using low energy near-infrared (NIR) light. The promising features of two-photon imaging in turn have inspired a quest for two-photon active fluorophores and probes.
Herein, several strategies for two-photon bioimaging has been explored to tackle the limitations of the existing bioimaging materials. This fundamental research provides essential guidelines for the development of novel two-photon materials/fluorophores with high emission intensity (Chem. Sci., 2015, 6, 4335) as well as emission at long wavelength to avoid the intrinsic autofluorescence from biomolecules (J. Am. Chem. Soc. 2015, 137, 6781; Chem. Commun., 2012, 48, 10243; Chem. Sci., 2019, 10, 9028). Accordingly, those newly developed materials act as excellent contrast agents in two-photon bioimaging.