3D sub-cellular localization of upconverting nanoparticles through hyperspectral microscopy

Hyperspectral microscopy is an intriguing technique combining spectroscopy with optical microscopy that can be used to simultaneously obtain spectral and spatial information. The relevance of hyperspectral imaging in biomedical applications such as the monitoring of bioimaging agents, the identification of pathogens and cancerous cells, and the cellular uptake of nanoparticles has emerged recently, due to recent advances in optical reconstruction. The location and tracking of particles within the cell structure have been analyzed by 2D hyperspectral imaging of non-fluorescence objects, being examples of 3D localization uncommon. Here, we report the synthesis of Yb³⁺/Er³⁺-codoped Gd₂O₃ nanoparticles, their structural and luminescence characterization, and their biocompatibility assessments in Human melanoma (MNT-1 and A375) cell lines. The internalization of the particles by MNT-1 cells and their 3D localization in a fixed configuration are addressed through 2D optical images acquired in different planes along with the cell culture depth. 2D hyperspectral imaging is used to unequivocally identify the nuclei and the nanoparticles. The results indicate that the particles are distributed in distinct planes deep in the cell volume in the cytoplasmic and perinuclear regions. Furthermore, the emission signature of the nanoparticles enabled the determination of the intracellular temperature.