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Proceedings Paper

ns-time resolution for multispecies STED-FLIM and artifact free STED-FCS
Author(s): Marcelle Koenig; Paja Reisch; Rhys Dowler; Benedikt Kraemer; Sebastian Tannert; Matthias Patting; Mathias P. Clausen; Silvia Galiani; Christian Eggeling; Felix Koberling; Rainer Erdmann
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Paper Abstract

Stimulated Emission Depletion (STED) Microscopy has evolved into a well established method offering optical superresolution below 50 nm. Running both excitation and depletion lasers in picosecond pulsed modes allows for highest optical resolution as well as fully exploiting the photon arrival time information using time-resolved single photon counting (TCSPC). Non-superresolved contributions can be easily dismissed through time-gated detection (gated STED) or a more detailed fluorescence decay analysis (FLIM-STED), both leading to an even further improved imaging resolution. Furthermore, these methods allow for accurate separation of different fluorescent species, especially if subtle differences in the excitation and emission spectra as well as the fluorescence decay are taken into account in parallel. STED can also be used to shrink the observation volume while studying the dynamics of diffusing species in Fluorescence Correlation Spectroscopy (FCS) to overcome averaging issues along long transit paths. A further unique advantage of STED-FCS is that the observation spot diameter can be tuned in a gradual manner enabling, for example, determining the type of hindered diffusion in lipid membrane studies. Our completely pulsed illumination scheme allows realizing an improved STED-FCS data acquisition using pulsed interleaved excitation (PIE). PIE-STED-FCS allows for a straightforward online check whether the STED laser has an influence on the investigated diffusion dynamics.

Paper Details

Date Published: 14 March 2016
PDF: 9 pages
Proc. SPIE 9712, Multiphoton Microscopy in the Biomedical Sciences XVI, 97120T (14 March 2016); doi: 10.1117/12.2208874
Show Author Affiliations
Marcelle Koenig, PicoQuant GmbH (Germany)
Paja Reisch, PicoQuant GmbH (Germany)
Rhys Dowler, PicoQuant GmbH (Germany)
Benedikt Kraemer, PicoQuant GmbH (Germany)
Sebastian Tannert, PicoQuant GmbH (Germany)
Matthias Patting, PicoQuant GmbH (Germany)
Mathias P. Clausen, Weatherall Institute of Molecular Medicine, Univ. of Oxford (United Kingdom)
Silvia Galiani, Weatherall Institute of Molecular Medicine, Univ. of Oxford (United Kingdom)
Christian Eggeling, Weatherall Institute of Molecular Medicine, Univ. of Oxford (United Kingdom)
Felix Koberling, PicoQuant GmbH (Germany)
Rainer Erdmann, PicoQuant GmbH (Germany)


Published in SPIE Proceedings Vol. 9712:
Multiphoton Microscopy in the Biomedical Sciences XVI
Ammasi Periasamy; Peter T. C. So; Karsten König, Editor(s)

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