title: Spectrally coded optical nanosectioning (SpecON) with biocompatible metal-dielectric-coated substrates.
abstract: Fluorescence nanosectioning within a submicron region above an interface is desirable for many disciplines in the life sciences. A drawback, however, to most current approaches is the a priori need to physically scan a sculptured point spread function in the axial dimension, which can be undesirable for optically sensitive or highly dynamic samples. Here we demonstrate a fluorescence imaging approach that can overcome the need for scanning by exploiting the position-dependent emission spectrum of fluorophores above a simple biocompatible nanostructure. To achieve this we have designed a thin metal-dielectric-coated substrate, where the spectral modification to the total measured fluorescence can be used to estimate the axial fluorophore distribution within distances of 10-150 nm above the substrate with an accuracy of up to 5-10 nm. The modeling and feasibility of the approach are verified and successfully applied to elucidate nanoscale adhesion protein and filopodia dynamics in migrating cells. It is likely that the general principle can find broader applications in, for example, single-molecule studies, biosensing, and studying fast dynamic processes.
authors.affiliation: Research Institute of Molecular Pathology, 1030 Vienna, Austria.
authors.forename: Kareem
authors.initials: K
authors.lastname: Elsayad
authors.forename: Alexander
authors.initials: A
authors.lastname: Urich
authors.forename: Piau Siong
authors.initials: PS
authors.lastname: Tan
authors.forename: Maria
authors.initials: M
authors.lastname: Nemethova
authors.forename: J Victor
authors.initials: JV
authors.lastname: Small
authors.forename: Karl
authors.initials: K
authors.lastname: Unterrainer
authors.forename: Katrin G
authors.initials: KG
authors.lastname: Heinze
mesh_terms: D002465:Cell Movement / Q000502:physiology*; D031541:Fluorescence Resonance Energy Transfer; D008670:Metals / Q000737:chemistry*; D008853:Microscopy / Q000379:methods; D008867:Microtomy / Q000379:methods*; D008962:Models, Theoretical; D049329:Nanostructures
chemical_list: D008670:Metals
keywords: cell adhesions; fluorescence microscopy; sectioning; superresolution