An efficient two-photon ratiometric fluorescent probe platform for dual-channel imaging of lysosomes in living cells and tissues

In contrast to one-photon microscopy, two-photon probe-based fluorescence imaging can provide improved three-dimensional spatial localization and increased imaging depth. Therefore, the development of new functional two-photon fluorescent dye has attracted great attention. Herein, we have adopted the fluorescence resonance energy transfer (FRET) strategy to design a unique type of two photon ratiometric fluorescent probe platform. Specifically, a two-photon fluorophore (D-pi-A-structured naphthalimide derivative) and a rhodamine B fluorophore are directly connected by a flexible piperidine linker. We further used this platform to develop a new type of two-photon ratiometric fluorescent probe, NRLys, for lysosomal pH detection and bioimaging. The experiments demonstrated that the NRLys is a reliable and specific probe for labeling lysosomes in living cells and tissues with two well-resolved emission peaks separated by 60 nm. The probe showed high ratiometric imaging resolution and deep-tissue imaging depth of over 180 mu m. Based on the above results, we expect that the new platform may prompt the development of a wide variety of two-photon ratiometric fluorescent probes application in biological systems. (C) 2016 Elsevier B.V. All rights reserved.