Two-Photon Upconversion Laser (Scanning and Wide-Field) Microscopy Using Ln3+-Doped NaYF4 Upconverting Nanocrystals: A Critical Evaluation of their Performance and Potential in Bioimaging

We report a simple, yet effective method to disperse NaYF4 nanocrystals (NCs) doped with luminescent Ln(3+) ions in water and physiological buffers using an amphiphilic polymer poly(ethylene glycol) monooleate. These water-dispersible NCs were used for in vivo imaging by employing two-photon upconversion laser scanning microscopy (TPULSM) and two-photon upconversion wide field microscopy (TPUWFM) techniques. Using the 800 nm upconverted emission from Tm3+ ions, we show that (i) TPULSM imaging can be performed up to a depth of similar to 600 mu m inside an agar-milk gel tissue phantom and (ii) the edges of the object can still be identified. At depths beyond 600 mu m, we observed a drastic decrease in the lateral resolution. Images of a mouse lung tissue obtained using this technique resulted in a lateral resolution with which we could observe the capillaries surrounding the alveoli air caps. The images lacked optical sectioning due to the high power density (similar to 2000 W/cm(2)) necessary to achieve an adequate signal-to-noise ratio. In addition, the time taken to obtain these images was prolonged because of the slow scanning speed necessitated by the long lifetimes and the poor quantum yield of the upconversion process. Conversely, in vivo TPUWFM imaging using the same 800 nm emission of brain blood vessels of a mouse after skull thinning gave excellent lateral resolution to differentiate blood vessels separated by a few micrometers. In addition to this, optical sectioning was observed over a depth of 100 mu m, which is the first instance of optical sectioning shown in in vivo imaging employing Ln(3+)-doped NCs as imaging agents. Experiments with the aforementioned tissue phantom showed that imaging up to a depth of similar to 400 mu m could be obtained with the 800 nm emission from Tm3+/Yb3+ codoped NaYF4 NCs with a lateral resolution that allows us to distinguish micrometer-sized biological structures. In contrast, when employing the green upconverted emission from Er3+/Yb3+ codoped NaYF4 NCs, lateral resolution was completely lost at a depth of similar to 300 mu m.