Rational Design of Cyclometalated Iridium(III) Complexes for Three-Photon Phosphorescence Bioimaging.

Compared to two-photon excitation (2PE) microscopy, three-photon excitation (3PE) microscopy has superior spatial resolution, deeper tissue penetration, and less defocused interference, and is, therefore, of great interest in the field of bioimaging. The design of suitable agents which can meet the various needs of the technology, such as large Stokes shift, good three-photon absorption (3PA), subcellular targeting, and fluorescence lifetime imaging (FLIM) properties, is a formidable challenge. In order to address this, two iridium(III) complexes (3PAIr1 and 3PAIr2) were developed as efficient three-photon phosphorescence (3PP) agents for bioimaging. Theoretical calculations reveal that the introduction of a new group to the molecular scaffold confers a quadruple promotion in three-photon transition probability. Herein, the confocal and lifetime imaging of mitochondria as using Ir(III) complexes as 3PP agents is demonstrated for the first time. The complexes exhibit the advantages of low working concentration (50 nM), fast uptake (5 min), and low threshold for three-photon excitation power (ca. 0.5 mW at 980 nm). Moreover, the impressive tissue penetration depth achieved with these 3PA agents (ca. 450 µm) allowed the 3D imaging and reconstruction of brain vasculature from a living specimen. These results indicate that the Ir(III) complexes are highly promising for medical imaging applications.