Organic dye doped nanoparticles with NIR emission and biocompatibility for ultra-deep in vivo two-photon microscopy under 1040 nm femtosecond excitation

Nanoparticle-assisted near-infrared (NIR) bioimaging and two-photon fluorescence microscopy (TPFM) are two important technologies in biophotonic research. In this work, we synthesize the dye named 2-(4-bromophenyl)-3-(4-(4-(diphenylamino)styryl)phenyl)fumaronitrile (TPABDFN), which had a large two-photon absorption cross-section and bright NIR emission. The dyes were then encapsulated with poly(styrene-co-maleic anhydride) (PSMA), forming fluorescent nanoparticles. The TPABDFN-PSMA nanoparticles possessed high chemical and optical stability, good biocompatibility, as well as large two-photon absorption cross-section (5.56 × 10⁵ GM). Furthermore, we combined NIR bioimaging and TPFM together, and utilized TPABDFN-PSMA nanoparticles as fluorescent contrast agents for two-photon excited NIR microscopic imaging, with a 1040 nm-femtosecond laser. In vivo angiography of mice ear and brain was performed. Due to the deep penetration capability of both 1040 nm-excitation and NIR emission light, a very large in vivo microscopic imaging depth (∼1.2 mm) was achieved. NIR emissive and biocompatible TPABDFN-PSMA nanoparticles have great potential in disease diagnosis and clinical therapies, where deep-tissue imaging is required.