High-Fidelity Hydrophilic Probe for Two-Photon Fluorescence Lysosomal Imaging

[Image: see text] The synthesis and characterization of a novel two-photon-absorbing fluorene derivative, LT1, selective for the lysosomes of HCT 116 cancer cells, is reported. Linear and nonlinear photophysical and photochemical properties of the probe were investigated to evaluate the potential of the probe for two-photon fluorescence microscopy (2PFM) lysosomal imaging. The cytotoxicity of the probe was investigated to evaluate the potential of using this probe for live two-photon fluorescence biological imaging applications. Colocalization studies of the probe with commercial Lysotracker Red in HCT 116 cells demonstrated the specific localization of the probe in the lysosomes with an extremely high colocalization coefficient (0.96). A figure of merit was introduced to allow comparison between probes. LT1 has a number of properties that far exceed those of commercial lysotracker probes, including higher two-photon absorption cross sections, good fluorescence quantum yield, and, importantly, high photostability, all resulting in a superior figure of merit. 2PFM was used to demonstrate lysosomal tracking with LT1.