Scanning two-photon microscopy with upconverting lanthanide nanoparticles via Richardson-Lucy deconvolution.

The use of upconverting lanthanide nanoparticles in fast-scanning microscopy is hindered by a long luminescence decay time, which greatly blurs images acquired in a nondescanned mode. We demonstrate herein an image processing method based on Richardson-Lucy deconvolution that mitigates the detrimental effects of their luminescence lifetime. This technique generates images with lateral resolution on par with the system's performance, ∼1.2  μm, while maintaining an axial resolution of 5 μm or better at a scan rate comparable with traditional two-photon microscopy. Remarkably, this can be accomplished with near infrared excitation power densities of 850 W/cm(2), several orders of magnitude below those used in two-photon imaging with molecular fluorophores. By way of illustration, we introduce the use of lipids to coat and functionalize these nanoparticles, rendering them water dispersible and readily conjugated to biologically relevant ligands, in this case epidermal growth factor receptor antibody. This deconvolution technique combined with the functionalized nanoparticles will enable three-dimensional functional tissue imaging at exceptionally low excitation power densities.