A ratiometric near-infrared fluorescence strategy based on spiropyran in situ switching for tracking dynamic changes of live-cell lysosomal pH

Abstract

Abnormal lysosomal pH values have been associated with cellular dysfunctions and many diseases. However, tools to monitor dynamic changes of pH values in live-cell lysosomes are limited. Herein, biocompatible near-infrared (NIR) fluorescent dyes HAN1-4 for lysosome-targeting staining were rationally designed and synthesized with the aid of a spiropyran in situ switching (SIS) strategy. The novel SIS-based dyes share the following special characteristics: 1) pH-dependent ratiometric fluorescent patterns matched nicely with pH windows of lysosomes, 2) light-independent real-time spiropyran in situ switching behavior in lysosomes and 3) pH-sensitive fluorescence in the NIR region, thereby ensuring their applications in obtaining highly specific images of live-cell lysosomes with a weak background signal. Among them, the HAN2 probe was found to induce the best ratiometric response toward pH in the range corresponding to lysosomal pH window, thus being further applied for the real-time tracking of lysosomal subtle pH changes in stressed cells undergoing drug and heat stimulations. Live-cell super-resolution and confocal fluorescent imaging indicate that our SIS-based NIR probes are robust and can be applied for the accurate detection of lysosomal pH in complex biosystems.