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Visualization of intracellular pH (i-pH) using surface-enhanced Raman spectroscopy (SERS) plays an important role toward understanding of cellular processes including their interactions with nanoparticles. However, conventional two-dimensional SERS imaging often fails to take into consideration changes occurring in the whole-cell volume. We therefore aimed at obtaining a comprehensive i-pH profile of living cells by means of three-dimensional (3D) SERS imaging, thereby visualizing dynamic i-pH distribution changes in a single cell. We devised here a biocompatible and highly stable SERS pH probe, comprising plasmonic gold nanostars functionalized with a pH-sensitive Raman reporter tag-4-mercaptobenzoic acid-and protected by a cationic biocompatible polymer, poly-l-arginine hydrochloride (PA). The positively charged PA coating plays a double role in enhancing cell uptake and providing chemical and colloidal stability in cellular environments. The SERS-active pH probe allowed visualization of local changes in i-pH, such as acidification during nanoparticle (NP) endocytosis. We provide evidence of i-pH changes during NP endocytosis via high-resolution 3D SERS imaging, thereby opening new avenues toward the application of SERS to intracellular studies.

Live-Cell Surface-Enhanced Raman Spectroscopy Imaging of Intracellular pH: From Two Dimensions to Three Dimensions