Illuminating the unknown: Development of emission-ratiometric, Zn(II)-selective fluorescent probes for two-photon excitation microscopy

Abstract

Zinc plays critical roles as an essential trace micronutrient in biology by participating in multifarious biological processes that include protein synthesis, gene regulation, metabolic reactions, and DNA repair. While the majority of intracellular Zn(II) is tightly bound to endogenous proteins, there is growing evidence that cells regulate a buffered, kinetically labile pool of Zn(II). At present, the mechanisms that control Zn(II) regulation and distribution during cell growth and development largely remain unexplored, in part due to the challenges associated with phototoxicity and photobleaching of fluorescent probes that are used as indicators for detecting labile Zn(II) when utilized over extended periods of time. Providing an attractive solution over conventional linear optics, two-photon excitation microscopy (TPEM) allows fluorophores to be excited in the near-infrared region, thus offering improved depth penetration combined with reduced phototoxicity and photobleaching, as only fluorophores in the focal plane are excited. Take advantage of TPEM, a series of emission-ratiometric two-photon-excitable fluorescent probes that selectively bind Zn(II) with a 1:1 stoichiometry was developed and optimized for TPEM. Designed based on a non-centrosymmetric donor-π-acceptor architecture, the inherent fluorophore platform results in a balanced two photon cross section and a significant chromatic shift upon saturation with Zn(II). Using these optimized probes, the dynamics of labile Zn(II) pools in proliferating 3T3 mouse fibroblast cells using live-cell TPEM were investigated, with potential application in higher organisms, such as zebrafish and mice.