Abstract
Abstract We present localization with stimulated emission depletion (LocSTED) microscopy, a combination of STED and single-molecule localization microscopy (SMLM). We use the simplest form of a STED microscope that is cost effective and synchronization free, comprising continuous wave (CW) lasers for both excitation and depletion. By utilizing the reversible blinking of fluorophores, single molecules of Alexa 555 are localized down to ~5 nm. Imaging fluorescently labeled proteins attached to nanoanchors structured by STED lithography shows that LocSTED microscopy can resolve molecules with a resolution of at least 15 nm, substantially improving the classical resolution of a CW STED microscope of about 60 nm. LocSTED microscopy also allows estimating the total number of proteins attached on a single nanoanchor.
Highlights
For more than a century, the diffraction limit of light was considered to be the major barrier in optical microscopy according to Abbe [1]
We present localization with stimulated emission depletion (LocSTED) microscopy, a combination of STED and single-molecule localization microscopy (SMLM)
Imaging fluorescently labeled proteins attached to nanoanchors structured by STED lithography shows that LocSTED microscopy can resolve molecules with a resolution of at least 15 nm, substantially improving the classical resolution of a continuous wave (CW) STED microscope of about 60 nm
Summary
For more than a century, the diffraction limit of light was considered to be the major barrier in optical microscopy according to Abbe [1]. In STED microscopy and related techniques, one uses the spatial depletion of molecules such that fluorescence occurs only within a predefined, nanoscopic spatial area. This is achieved by overlapping a regularly focused excitation beam with a red-shifted “STED beam” or “depletion beam” in the shape of a donut with a zero central intensity.
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