A STED-FLIM microscope applied to imaging the natural killer cell immune synapse

M. O. Lenz; A. C. N. Brown; E. Auksorius; D. M. Davis; C. Dunsby; M. A. A. Neil; P. M. W. French

doi:10.1117/12.875018

28 February 2011 A STED-FLIM microscope applied to imaging the natural killer cell immune synapse

M. O. Lenz, A. C. N. Brown, E. Auksorius, D. M. Davis, C. Dunsby, M. A. A. Neil, P. M. W. French

Author Affiliations +

Proceedings Volume 7903, Multiphoton Microscopy in the Biomedical Sciences XI; 79032D (2011) https://doi.org/10.1117/12.875018
Event: SPIE BiOS, 2011, San Francisco, California, United States

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.

Citation Download Citation

M. O. Lenz, A. C. N. Brown, E. Auksorius, D. M. Davis, C. Dunsby, M. A. A. Neil, and P. M. W. French "A STED-FLIM microscope applied to imaging the natural killer cell immune synapse", Proc. SPIE 7903, Multiphoton Microscopy in the Biomedical Sciences XI, 79032D (28 February 2011); https://doi.org/10.1117/12.875018

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