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 Ln³⁺ 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 ∼600 μm inside an agar-milk gel tissue phantom and (ii) the edges of the object can still be identified. At depths beyond 600 μm, 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 (∼2000 W/cm²) 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 μm, which is the first instance of optical sectioning shown in in vivo imaging employing Ln³⁺-doped NCs as imaging agents. Experiments with the aforementioned tissue phantom showed that imaging up to a depth of ∼400 μm 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 ∼300 μm.