An effective strategy is presented to make spherical Ln3+ doped NaYF4 nanoparticles that show upconversion, with the
aim of deep-tissue optical imaging. Upconversion is the conversion of two or more low-energy photons into one of
higher energy, e.g. 980 nm to 545 and 680 nm and 980 nm to 800 nm. In order to avoid the formation of nanoparticles
with an aspect ratio, we developed a strategy in which subsequent shells were grown on spherical seed nanoparticles.
The last shell is undoped in order to improve the optical properties. In addition, a simple intercalation strategy involving
the oleate ligands on the surface has been developed to make the nanoparticles dispersible in aqueous solutions and
physiological buffers. Two-photon upconversion laser scanning microscopy (TPULSM) and two-photon upconversion
wide-field microscopy (TPUWFM) have been tested for their suitability in deep-tissue imaging with retention of lateral
and depth resolution (also called optical sectioning). TPULSM can be used up to ~ 600 μm deep, but takes inordinately
long times to acquire, which is due to the fact that the absorption cross section of Yb3+ is low, the quantum yield of the
upconversion process are << 1%, and the Ln3+ excited states are up to several hundreds of μs. Hence UCNPs in general are not very bright (i.e. large emitted photon flux). The TPUWFM seems more promising because acquisition times are
only several minutes, with depth profiling up to 400 μm. We show the first optical sectioning with this technique in the
brain of a mouse, through a thin shaved skull.