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

[Display omitted] •A novel two-photon ratiometric fluorescent probe platform was developed using a FRET strategy.•A FRET-based two-photon ratiometric fluorescent probe NRLys was developed to monitor pH change in lysosome.•NRLys possesses two well-resolved emission peaks separated by 60nm.•The NRLys shows high ratiometric imaging resolution and deep-tissue imaging depth of over 180μm. 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-π-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 60nm. The probe showed high ratiometric imaging resolution and deep-tissue imaging depth of over 180μm. 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.