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Proceedings Paper

A STED-FLIM microscope applied to imaging the natural killer cell immune synapse
Author(s): M. O. Lenz; A. C. N. Brown; E. Auksorius; D. M. Davis; C. Dunsby; M. A. A. Neil; P. M. W. French
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Paper Abstract

We present a stimulated emission depletion (STED) fluorescence lifetime imaging (FLIM) microscope, excited by a microstructured optical fibre supercontinuum source that is pumped by a femtosecond Ti:Sapphire-laser, which is also used for depletion. Implemented using a piezo-scanning stage on a laser scanning confocal fluorescence microscope system with FLIM realised using time correlated single photon counting (TCSPC), this provides convenient switching between confocal and STED-FLIM with spatial resolution down to below 60 nm. We will present our design considerations to make a robust instrument for biological applications including a comparison between fixed phase plate and spatial light modulator (SLM) approaches to shape the STED beam and the correlation of STED and confocal FLIM microscopy. Following our previous application of FLIM-FRET to study intercellular signalling at the immunological synapse (IS), we are employing STED microscopy to characterize the spatial distribution of cellular molecules with subdiffraction resolution at the IS. In particular, we are imaging cytoskeletal structure at the Natural Killer cell activated immune synapse. We will also present our progress towards multilabel STED microscopy to determine how relative spatial molecular organization, previously undetectable by conventional microscopy techniques, is important for NK cell cytotoxic function. Keywords: STED, Stimulated Emission Depletion Microscopy, Natural Killer (NK) cell, Fluorescence lifetime imaging, FLIM, Super resolution microscopy.

Paper Details

Date Published: 28 February 2011
PDF: 6 pages
Proc. SPIE 7903, Multiphoton Microscopy in the Biomedical Sciences XI, 79032D (28 February 2011); doi: 10.1117/12.875018
Show Author Affiliations
M. O. Lenz, Imperial College London (United Kingdom)
A. C. N. Brown, Imperial College London (United Kingdom)
E. Auksorius, Imperial College London (United Kingdom)
D. M. Davis, Imperial College London (United Kingdom)
C. Dunsby, Imperial College London (United Kingdom)
M. A. A. Neil, Imperial College London (United Kingdom)
P. M. W. French, Imperial College London (United Kingdom)

Published in SPIE Proceedings Vol. 7903:
Multiphoton Microscopy in the Biomedical Sciences XI
Ammasi Periasamy; Karsten König; Peter T. C. So, Editor(s)

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