Cryo-Electron Microscopy and Confocal Microscopy to Study the Pathway of Ultrasmall Gold Nanoparticles Inside Cells

The cellular uptake and intracellular distribution of fluorescently labeled ultrasmall gold nanoparticles (∼2 nm) in human RPE-1 cells were investigated by a complementary combination of confocal laser scanning microscopy (CLSM), structured illumination microscopy (SIM), and cryo-electron microscopy (cryo-EM). The nanoparticles were synthesized by a modified Brust−Schiffrin route, functionalized with glutathione, and covalently labeled with AlexaFluor-647, enabling fluorescence-based tracking. High-resolution transmission electron microscopy showed a narrow particle size distribution with an average core diameter of 2.3 nm. Live-cell CLSM and SIM imaging revealed intracellular nanoparticle-associated fluorescence and pronounced colocalization with lysosomes, consistent with accumulation along the endolysosomal pathway. SIM provided an enhanced spatial resolution compared to CLSM, allowing a more detailed visualization of the intracellular nanoparticle localization. Notably, in addition to endolysosomal localization, ultrasmall gold nanoparticles were occasionally observed inside the cell nucleus. Correlative light and cryo-electron tomography further enabled direct visualization of the electron-dense gold nanoparticles within vitrified cellular lamellae at near-native conditions. Some nanoparticles were found inside a multi-vesicular body, several large clusters of nanoparticles in a functional lysosome, and single nanoparticles and clusters in a late endosome or early lysosome. No nanoparticles were observed inside the nucleus by cryo-EM.