We demonstrate stimulated emission depletion (STED) microscopy implemented in a laser scanning confocal microscope using excitation light derived from supercontinuum generation in a microstructured optical fiber. Images with resolution improvement beyond the far-field diffraction limit in both the lateral and axial directions were acquired by scanning overlapped excitation and depletion beams in two dimensions using the flying spot scanner of a commercially available laser scanning confocal microscope. The spatial properties of the depletion beam were controlled holographically using a programmable spatial light modulator, which can rapidly change between different STED imaging modes and also compensate for aberrations in the optical path. STED fluorescence lifetime imaging microscopy is demonstrated through the use of time-correlated single photon counting.

We report the supramolecular organization of killer Ig–like receptor (KIR) phosphorylation using a technique applicable to imaging phosphorylation of any green fluorescent protein–tagged receptor at an intercellular contact or immune synapse. Specifically, we use fluorescence lifetime imaging (FLIM) to report Förster resonance energy transfer (FRET) between GFP-tagged KIR2DL1 and a Cy3-tagged generic anti-phosphotyrosine monoclonal antibody. Visualization of KIR phosphorylation in natural killer (NK) cells contacting target cells expressing cognate major histocompatibility complex class I proteins revealed that inhibitory signaling is spatially restricted to the immune synapse. This explains how NK cells respond appropriately when simultaneously surveying susceptible and resistant target cells. More surprising, phosphorylated KIR was confined to microclusters within the aggregate of KIR, contrary to an expected homogeneous distribution of KIR signaling across the immune synapse. Also, yellow fluorescent protein–tagged Lck, a kinase important for KIR phosphorylation, accumulated in a multifocal distribution at inhibitory synapses. Spatial confinement of receptor phosphorylation within the immune synapse may be critical to how activating and inhibitory signals are integrated in NK cells.

W e report the supramolecular organization of killer Ig-like receptor (KIR) phosphorylation using a technique applicable to imaging phosphorylation of any green fl uorescent protein-tagged receptor at an intercellular contact or immune synapse. Specifi cally, we use fl uorescence lifetime imaging (FLIM) to report Förster resonance energy transfer (FRET) between GFP-tagged KIR2DL1 and a Cy3-tagged generic anti-phosphotyrosine monoclonal antibody. Visualization of KIR phosphorylation in natural killer (NK) cells contacting target cells expressing cognate major histocompatibility complex class I proteins revealed that inhibitory signaling is spatially restricted to the immune synapse.This explains how NK cells respond appropriately when simultaneously surveying susceptible and resistant target cells. More surprising, phosphorylated KIR was confi ned to microclusters within the aggregate of KIR, contrary to an expected homogeneous distribution of KIR signaling across the immune synapse. Also, yellow fl uorescent protein-tagged Lck, a kinase important for KIR phosphorylation, accumulated in a multifocal distribution at inhibitory synapses. Spatial confi nement of receptor phosphorylation within the immune synapse may be critical to how activating and inhibitory signals are integrated in NK cells.Correspondence to Daniel M. Davis: d.davis@imperial.ac.uk Abbreviations used in this paper: FLIM, fl uorescence lifetime imaging; FRET, Förster resonance energy transfer; HLA, human leukocyte antigen; IS, immunological synapse; ITIM, immunoreceptor tyrosine-based inhibition motif; KIR, killer Ig-like receptor; MHC, major histocompatibility complex; mYFP, monomeric YFP; NK, natural killer; SHP, Src homology protein tyrosine phosphatase.

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