Journal
JOURNAL OF BIOPHOTONICS
Volume 7, Issue 6, Pages 376-380Publisher
WILEY-V C H VERLAG GMBH
DOI: 10.1002/jbio.201300208
Keywords
fluorescence microscopy; stimulated emission depletion (STED) microscopy; super-resolution microscopy; background subtraction
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Funding
- Italian Ministry of Education, University and Research (MIUR) through PRIN project [2008S22MJC 005]
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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. [GRAPHICS] Principle and time course of the measurement for the proposed anti-Stokes emission background subtraction method (right). Application of the method for a fluorescent beads calibration sample (left).
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