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

Abstract

Abstract. 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 threedimensional functional tissue imaging at exceptionally low excitation power densities.

Multiphoton imaging of upconverting lanthanide nanoparticles in three dimensional models of cancer

“…In order to render UNPs hydrophilic and provide surface groups for functionalization, we coat UNPs with a lipid monolayer. 5,6 …”

A review of synthetic methods for the production of upconverting lanthanide nanoparticles

“…Functionalization with maleimide has also been proposed recently as a convenient method for the attachment of proteins. 70 For simplicity, surface modi¯cation methods investigated so far can be roughly divided into¯ve categories: ligand exchange, 55,[71][72][73][74] ligand modi¯cation, 75,76 ligand removal, 52 adsorption of amphiphilic molecules 4,5,[77][78][79][80] and growth of a silica shell. 81,82 From our experience, coating with amphiphilic molecules is the simplest surface modi¯cation method both for dispersing UCNPs in aqueous solutions and for functionalization with biologically relevant ligands.…”

Poor man's two photon imaging: Scanning Laser Upconversion Microscopy

“…This is a hard task to accomplish due to the long characteristic times of UCNP emission, which implies very low scanning sweeping speed or image smearing. Romanowsky have devised a clever procedure to reconstruct smeared images through deconvolution, useful for sparse labelling 15 , while Pierce have attacked the problem by means of line confocal microscopy 16 , where just the slow axis needs to be swept. However, these latter approaches also allow image sectioning by means of confocal procedures, not using the intrinsic sectioning power of upconversion as a nonlinear emission process.…”