Single-particle luminescence microscopy is a powerful method to extract information on biological systems that is not accessible by ensemble-level methods. Upconversion nanoparticles (UCNPs) are highly suited for single-particle microscopy, as they provide stable, non-blinking luminescence, and avoid biological autofluorescence by their anti-Stokes emission. Recently, ensemble measurements of diluted aqueous dispersions of UCNPs have shown an instability of luminescence over time due to particle dissolution-related effects. This can be especially detrimental for single-particle experiments. However, this effect has never been estimated at the individual particle level. Here, the luminescence response of individual UCNPs in aqueous conditions is investigated by quantitative wide-field microscopy. The particles exhibit a rapid luminescence loss, accompanied by large changes in spectral response, leading to a considerable heterogeneity in their luminescence and band intensity ratio. Moreover, the dissolution-caused intensity loss is not correlated with initial particle intensity or band ratio, which makes it virtually unpredictable. These effects and the subsequent development of their heterogeneity can be largely slowed down by adding millimolar concentrations of sodium fluoride in the buffer. As a consequence, our data indicate that single molecule microscopy experiments employing UCNPs in aqueous environment should be performed in conditions that carefully prevent these effects.

Date Published: 13 March 2019
PDF
Proc. SPIE 10884, Single Molecule Spectroscopy and Superresolution Imaging XII, 108840V (13 March 2019); doi: 10.1117/12.2511077

Published in SPIE Proceedings Vol. 10884:
Single Molecule Spectroscopy and Superresolution Imaging XII
Zygmunt Karol Gryczynski; Ingo Gregor; Felix Koberling, Editor(s)