Upconversion luminescence imaging of cells and small animals

Upconversion luminescence (UCL) is an anti-Stokes process whereby low-energy photons are converted to higher-energy ones. UCL imaging for cells and animal tissues has attracted substantial attention in recent years because of the unique abilities of upconversion materials, which can minimize the background interference from the autofluorescence of biosamples and enhance tissue penetration. This protocol describes a step-by-step guide for the fabrication of UCL probes, including lanthanide-based upconversion nanoparticles (Ln-UCNPs) with a particle size of ∼20 nm (NaYF4/NaLuF4: Yb, Er/Tm) and triplet-triplet annihilation-based UCNPs (TTA-UCNPs) with a particle size of ∼10 nm (palladium octaethylporphyrin as sensitizer and 9,10-diphenylanthracene as annihilator). We also describe the characterization of the UCL nanoprobes (via transmission electron microscopy and UCL emission spectroscopy) and functionalization (via silica coating and ligand exchange), as well as applications for UCL bioimaging of living cells (HeLa cells) and small animals (nude mice and Kunming mice). The setup of a laser-scanning UCL microscope and a UCL imaging system is also presented. Compared with a normal imaging setup, we adopted longer-wavelength excitation lasers and short-pass filters. The synthesis of hydrophilic UCNP for application in UCL bioimaging requires ∼15 d.