Paul M. W. French

Time-resolved fluorescence microscopy

In fluorescence microscopy, the fluorescence emission can be characterised not only by intensity and position, but also by lifetime, polarization and wavelength. Fluorescence lifetime imaging (FLIM) can report on photophysical events that are difficult or impossible to observe by fluorescence intensity imaging, and time-resolved fluorescence anisotropy imaging (TR-FAIM) can measure the rotational mobility of a fluorophore in its environment. We compare different FLIM methods: a chief advantage of wide-field time-gating and phase modulation methods is the speed of acquisition whereas for time-correlated single photon counting (TCSPC) based confocal scanning it is accuracy in the fluorescence decay. FLIM has been used to image interactions between proteins such as receptor oligomerisation and to reveal protein phosphorylation by detecting fluorescence resonance energy transfer (FRET). In addition, FLIM can also probe the local environment of fluorophores, reporting, for example, on the local pH, refractive index, ion or oxygen concentration without the need for ratiometric measurements.

Structurally Distinct Membrane Nanotubes between Human Macrophages Support Long-Distance Vesicular Traffic or Surfing of Bacteria

We report that two classes of membrane nanotubes between human monocyte-derived macrophages can be distinguished by their cytoskeletal structure and their functional properties. Thin membrane nanotubes contained only F-actin, whereas thicker nanotubes, i.e., those > ∼0.7 μm in diameter, contained both F-actin and microtubules. Bacteria could be trapped and surf along thin, but not thick, membrane nanotubes toward connected macrophage cell bodies. Once at the cell body, bacteria could then be phagocytosed. The movement of bacteria is aided by a constitutive flow of the nanotube surface because streptavidin-coated beads were similarly able to traffic along nanotubes between surface-biotinylated macrophages. Mitochondria and intracellular vesicles, including late endosomes and lysosomes, could be detected within thick, but not thin, membrane nanotubes. Analysis from kymographs demonstrated that vesicles moved in a stepwise, bidirectional manner at ∼1 μm/s, consistent with their traffic being mediated by the microtubules found only in thick nanotubes. Vesicular traffic in thick nanotubes and surfing of beads along thin nanotubes were both stopped upon the addition of azide, demonstrating that both processes require ATP. However, microtubule destabilizing agents colchicine or nocodazole abrogated vesicular transport but not the flow of the nanotube surface, confirming that distinct cytoskeletal structures of nanotubes give rise to different functional properties. Thus, membrane nanotubes between macrophages are more complex than unvarying ubiquitous membrane tethers and facilitate several means for distal interactions between immune cells.

Application of the stretched exponential function to fluorescence lifetime imaging.

Conventional analyses of fluorescence lifetime measurements resolve the fluorescence decay profile in terms of discrete exponential components with distinct lifetimes. In complex, heterogeneous biological samples such as tissue, multi-exponential decay functions can appear to provide a better fit to fluorescence decay data than the assumption of a mono-exponential decay, but the assumption of multiple discrete components is essentially arbitrary and is often erroneous. Moreover, interactions, both between fluorophores and with their environment, can result in complex fluorescence decay profiles that represent a continuous distribution of lifetimes. Such continuous distributions have been reported for tryptophan, which is one of the main fluorophores in tissue. This situation is better represented by the stretched-exponential function (StrEF). In this work, we have applied, for the first time to our knowledge, the StrEF to time-domain whole-field fluorescence lifetime imaging (FLIM), yielding both excellent tissue contrast and goodness of fit using data from rat tissue. We note that for many biological samples for which there is no a priori knowledge of multiple discrete exponential fluorescence decay profiles, the StrEF is likely to provide a truer representation of the underlying fluorescence dynamics. Furthermore, fitting to a StrEF significantly decreases the required processing time, compared with a multi-exponential component fit and typically provides improved contrast and signal/noise in the resulting FLIM images. In addition, the stretched-exponential decay model can provide a direct measure of the heterogeneity of the sample, and the resulting heterogeneity map can reveal subtle tissue differences that other models fail to show.

Continuous-Flow Polymerase Chain Reaction of Single-Copy DNA in Microfluidic Microdroplets

We present a high throughput microfluidic device for continuous-flow polymerase chain reaction (PCR) in water-in-oil droplets of nanoliter volumes. The circular design of this device allows droplets to pass through alternating temperature zones and complete 34 cycles of PCR in only 17 min, avoiding temperature cycling of the entire device. The temperatures for the applied two-temperature PCR protocol can be adjusted according to requirements of template and primers. These temperatures were determined with fluorescence lifetime imaging (FLIM) inside the droplets, exploiting the temperature-dependent fluorescence lifetime of rhodamine B. The successful amplification of an 85 base-pair long template from four different start concentrations was demonstrated. Analysis of the product by gel-electrophoresis, sequencing, and real-time PCR showed that the amplification is specific and the amplification factors of up to 5 x 10(6)-fold are comparable to amplification factors obtained in a benchtop PCR machine. The high efficiency allows amplification from a single molecule of DNA per droplet. This device holds promise for convenient integration with other microfluidic devices and adds a critical missing component to the laboratory-on-a-chip toolkit.

Imaging the environment of green fluorescent protein.

An emerging theme in cell biology is that cell surface receptors need to be considered as part of supramolecular complexes of proteins and lipids facilitating specific receptor conformations and distinct distributions, e.g., at the immunological synapse. Thus, a new goal is to develop bioimaging that not only locates proteins in live cells but can also probe their environment. Such a technique is demonstrated here using fluorescence lifetime imaging of green fluorescent protein (GFP). We first show, by time-correlated single-photon counting, that the fluorescence decay of GFP depends on the local refractive index. This is in agreement with the Strickler Berg formula, relating the Einstein A and B coefficients for absorption and spontaneous emission in molecules. We then quantitatively image, by wide-field time-gated fluorescence lifetime imaging, the refractive index of the environment of GFP. This novel approach paves the way for imaging the biophysical environment of specific GFP-tagged proteins in live cells.

Remodelling of Cortical Actin Where Lytic Granules Dock at Natural Killer Cell Immune Synapses Revealed by Super-Resolution Microscopy

Super-resolution 3D imaging reveals remodeling of the cortical actin meshwork at the natural killer cell immune synapse, which is likely to be important for secretion of lytic granules.

Stimulated emission depletion microscopy with a supercontinuum source and fluorescence lifetime imaging

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.

Fluorescence lifetime imaging with picosecond resolution for biomedical applications.

We describe a novel whole-field fluorescence lifetime imaging system, based on a time-gated image intensifier and a solid-state laser oscillator-amplifier, that images lifetime differences of less than 10 ps. This system was successfully applied to discrimination between biological tissue constituents.

Fluorescence lifetime imaging distinguishes basal cell carcinoma from surrounding uninvolved skin

Background  Fluorescence lifetime imaging (FLIM) is a novel imaging technique that generates image contrast between different states of tissue due to differences in fluorescence decay rates.

Dynamics of subsynaptic vesicles and surface microclusters at the immunological synapse.

Vesicles dynamically deliver the T cell adaptor protein LAT to sites of T cell signalosomes. Submariner Adaptor Engagement of the T cell antigen receptor (TCR) on the surface of a T cell with peptide-loaded major histocompatibility complex on the surface of an antigen-presenting cell occurs at a specialized contact point known as the immunological synapse (IS). Stimulation of the TCR triggers the activation of the proximal kinases Lck and ZAP-70, which leads to the formation at the IS of microclusters of kinases and adaptor molecules that are required for T cell signaling. Two of these adaptor molecules are the cytosolic protein SLP-76 and the plasma membrane–associated protein LAT. SLP-76 and LAT form distinct microclusters, but how they interact to propagate T cell signals is unclear (see the Perspective by Billadeau). Purbhoo et al. used imaging techniques to show that a fraction of LAT was enriched in intracellular vesicles that resided below the IS and that these vesicles moved to the IS, where they interacted with SLP76-containing microclusters. LAT-containing vesicles were corralled by TCR–ZAP-70 microclusters in the IS and were more mobile than the SLP-76 microclusters, but they slowed when they interacted with SLP-76. Cells that contained a mutant LAT that could not interact with SLP-76 (through the associated protein GADS) contained fewer SLP-76 microclusters, and LAT-containing vesicles interacted less with them. Together, these data suggest that vesicular LAT interacts with protein microclusters at the IS and contributes to the propagation of T cell signaling. Imaging studies have identified clusters of kinases and adaptor proteins that serve as centers of signaling at the contact points between T cells and antigen-presenting cells (APCs). Here, we report that the kinase ZAP-70 and the adaptor proteins LAT and SLP-76 accumulated in separate clusters at the interface between T cells and coverslips coated with a stimulatory antibody against CD3, a component of the T cell antigen receptor complex. A fraction of LAT was detected in motile vesicles that repeatedly moved to surface microclusters of SLP-76 and the adaptor protein GADS (growth factor receptor–bound protein–related adaptor downstream of Shc), where they exhibited decreased motility. LAT molecules in which the residues tyrosine 171 and tyrosine 191 (which are required for the binding of LAT to GADS) were mutated to phenylalanine did not dwell at clusters of SLP-76. At immunological synapses, LAT-containing vesicles also colocalized with microclusters of SLP-76, as detected in experiments in which laser tweezers were used to position T cell–APC conjugates vertically for high-resolution imaging. Phosphorylation of LAT was most prominent when vesicular LAT colocalized with SLP-76. Indeed, the abundance of phosphorylated LAT within a microcluster of SLP-76 was greatest in those clusters that had more recent interactions with LAT-containing vesicles. Finally, negative signals by the inhibitory receptor ILT2 disrupted the assembly of SLP-76–containing microclusters. Together, these data show that the movement of LAT-containing vesicles is linked to the organization of protein microclusters and suggest an important role for vesicular LAT in the SLP-76 signalosome.