Single-nanocrystal sensitivity achieved by enhanced upconversion luminescence

Upconversion nanocrystals convert infrared radiation to visible luminescence, and are promising for applications in biodetection1-3, bioimaging4-7, solar cells8-10 and three-dimensional display technologies8,9,11. Although the design of suitable nanocrystals has improved the performance of upconversion nanocrystals10,12-14, their emission brightness is limited by the low doping concentration of activator ions needed to avoid the luminescence quenching that occurs at high concentrations15,16. Here, we demonstrate that high excitation irradiance can alleviate concentration quenching in upconversion luminescence when combined with higher activator concentration, which can be increased from 0.5 mol% to 8 mol% Tm31 in NaYF4. This leads to significantly enhanced luminescence signals, by up to a factor of 70. By using such bright nanocrystals, we demonstrate remote tracking of a single nanocrystal with a microstructured optical-fibre dip sensor. This represents a sensitivity improvement of three orders of magnitude over benchmark nanocrystals such as quantum dots17. © 2013 Macmillan Publishers Limited. All rights reserved

Downscaling the analysis of complex transmembrane signaling cascades to closed attoliter volumes.

Cellular signaling is classically investigated by measuring optical or electrical properties of single or populations of living cells. Here we show that ligand binding to cell surface receptors and subsequent activation of signaling cascades can be monitored in single, (sub-)micrometer sized native vesicles with single-molecule sensitivity. The vesicles are derived from live mammalian cells using chemicals or optical tweezers. They comprise parts of a cell's plasma membrane and cytosol and represent the smallest autonomous containers performing cellular signaling reactions thus functioning like minimized cells. Using fluorescence microscopies, we measured in individual vesicles the different steps of G-protein-coupled receptor mediated signaling like ligand binding to receptors, subsequent G-protein activation and finally arrestin translocation indicating receptor deactivation. Observing cellular signaling reactions in individual vesicles opens the door for downscaling bioanalysis of cellular functions to the attoliter range, multiplexing single cell analysis, and investigating receptor mediated signaling in multiarray format