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A New Filtering Technique for Removing Anti-Stokes Emission Background in Gated CW-STED Microscopy

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.


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Coto Hernàndez I, Peres C, Cella Zanacchi F, d'Amora M, Christodoulou S, Bianchini P, Diaspro A and Vicidomini G:
A new filtering technique for removing anti-Stokes emission background in gated CW-STED microscopy

J. Biophoton. (2014); doi: 10.1002/jbio.201300208