Intracellular Plasmonic Probes Show Molecular Effects of a Lysosomal Fluorescent Dye

Abstract

The choice of optical labels for live-cell imaging requires a characterization of interactions with their biological environment. Here, we discuss alterations of the molecular composition and interactions in the endolysosomal pathway of living cells by the fluorescent dye for lysosome labeling LysoSensor Green (LSG) based on vibrational nanospectroscopy using surface-enhanced Raman scattering (SERS) and soft X-ray nanotomography (SXT). A connection between the accumulation of LSG and ultrastructural changes is found by SXT that suggests an impact of the dye on cellular function. The biochemical effects in the endolysosomal system are detailed by different SERS experiments using gold nanoparticles as plasmonic probes, one that includes endolysosomes of all maturation stages and another one probing exclusively the lysosomal stage as the target compartment of the fluorescent label. The differences in SERS signals between the cells exposed to LSG compared to their controls are related to changes in the biochemical environment of the gold nanoparticles in the endolysosomal structures. As indicated by the occurrence of specific signals assigned to the interaction of the nanoprobes with proteins, nucleic acids, lipids, and LSG, respectively, the presence of LSG mainly affects the activity of enzymes that are involved in the degradation of proteins and nucleic acids in the lysosomes. The molecular changes when endolysosomes of all maturation stages contribute to the spectrum further point to LSG affecting cellular function along the whole endolysosomal pathway. The proposed complementary approach for nanoprobe characterization can be used for the validation of existing optical labels in fluorescence and multimodal imaging methods.