title: A new filtering technique for removing anti-Stokes emission background in gated CW-STED microscopy.
abstract: Stimulated emission depletion (STED) microscopy is a prominent approach of super-resolution optical microscopy, which allows cellular imaging with so far unprecedented unlimited spatial resolution. The introduction of time-gated detection in STED microscopy significantly reduces the (instantaneous) intensity required to obtain sub-diffraction spatial resolution. If the time-gating is combined with a STED beam operating in continuous wave (CW), a cheap and low labour demand implementation is obtained, the so called gated CW-STED microscope. However, time-gating also reduces the fluorescence signal which forms the image. Thereby, background sources such as fluorescence emission excited by the STED laser (anti-Stokes fluorescence) can reduce the effective resolution of the system. We propose a straightforward method for subtraction of anti-Stokes background. The method hinges on the uncorrelated nature of the anti-Stokes emission background with respect to the wanted fluorescence signal. The specific importance of the method towards the combination of two-photon-excitation with gated CW-STED microscopy is demonstrated.
authors.affiliation: Nanophysics, Istituto Italiano di Tecnologia, Via Morego 30, 16136, Genoa, Italy; Department of Physics, University of Genoa, Via Dodecaneso 33, 16146 Genoa, Italy.
authors.forename: Ivàn
authors.initials: I
authors.lastname: Coto Hernàndez
authors.forename: Chiara
authors.initials: C
authors.lastname: Peres
authors.forename: Francesca
authors.initials: F
authors.lastname: Cella Zanacchi
authors.forename: Marta
authors.initials: M
authors.lastname: d'Amora
authors.forename: Sotirios
authors.initials: S
authors.lastname: Christodoulou
authors.forename: Paolo
authors.initials: P
authors.lastname: Bianchini
authors.forename: Alberto
authors.initials: A
authors.lastname: Diaspro
authors.forename: Giuseppe
authors.initials: G
authors.lastname: Vicidomini
mesh_terms: D005453:Fluorescence; D006367:HeLa Cells; D006801:Humans; D007091:Image Processing, Computer-Assisted / Q000379:methods*; D007834:Lasers; D008856:Microscopy, Fluorescence / Q000379:methods*
keywords: background subtraction; fluorescence microscopy; stimulated emission depletion (STED) microscopy; super-resolution microscopy