Peter M. P. Lanigan

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.

Microclusters of inhibitory killer immunoglobulin–like receptor signaling at natural killer cell immunological synapses

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.

Time-resolved fluorescence anisotropy imaging applied to live cells

We have developed a wide-field time-resolved imaging system to image quantitatively both the fluorescence lifetime and the rotational correlation time of a fluorophore. Using a polarization-resolved imager, we simultaneously image orthogonal polarization components of the fluorescence emission onto a time-gated intensified CCD. We demonstrate imaging of solvent viscosity variations through the rotational correlation time of fluorescein in a multiwell plate and apply this technique to probe the microviscosity in live cells.

High speed optically sectioned fluorescence lifetime imaging permits study of live cell signaling events

We present a time domain optically sectioned fluorescence lifetime imaging (FLIM) microscope developed for high-speed live cell imaging. This single photon excited system combines wide field parallel pixel detection with confocal sectioning utilizing spinning Nipkow disc microscopy. It can acquire fluorescence lifetime images of live cells at up to 10 frames per second (fps), permitting high-speed FLIM of cell dynamics and protein interactions with potential for high throughput cell imaging and screening applications. We demonstrate the application of this FLIM microscope to real-time monitoring of changes in lipid order in cell membranes following cholesterol depletion using cyclodextrin and to the activation of the small GTP-ase Ras in live cells using FRET.

Imaging fluorescence lifetime heterogeneity applied to GFP-tagged MHC protein at an immunological synapse

Fluorescence imaging of green fluorescent protein (GFP) may be used to locate proteins in live cells and fluorescence lifetime imaging (FLIM) may be employed to probe the local microenvironment of proteins. Here we apply FLIM to GFP‐tagged proteins at the cell surface and at an inhibitory natural killer (NK) cell immunological synapse (IS). We present a novel quantitative analysis of fluorescence lifetime images that we believe is useful to determine whether apparent FLIM heterogeneity is statistically significant. We observe that, although the variation of observed fluorescence lifetime of GFP‐tagged proteins at the cell surface is close to the expected statistical range, the lifetime of GFP‐tagged proteins in cells is shorter than recombinant GFP in solution. Furthermore the lifetime of GFP‐tagged major histocompatibility complex class I protein is shortened at the inhibitory NK cell IS compared with the unconjugated membrane. Following our previous work demonstrating the ability of FLIM to report the local refractive index of GFP in solution, we speculate that these lifetime variations may indicate local refractive index changes. This application of our method for detecting small but significant differences in fluorescence lifetimes shows how FLIM could be broadly useful in imaging discrete membrane environments for a given protein.

Multifocal multiphoton excitation and time correlated single photon counting detection for 3-D fluorescence lifetime imaging

The interaction of matter and light is one of the fundamental processes occurring in nature, and its most elementary form is realized when a single atom interacts with a single photon. Reaching this regime has been a major focus of research in atomic physics and quantum optics for several decades and enables fascinating applications such as 3-D fluorescence imaging. Here we report a multifocal multiphoton time-correlated single photon counting (TCSPC) fluorescence lifetime imaging (FLIM) microscope system that uses a 16 channel multi-anode PMT detector. Multiphoton excitation minimizes out-of-focus photobleaching, multifocal excitation reduces non-linear in-plane photobleaching effects and TCSPC electronics provide photon-efficient detection of the fluorescence decay profile. TCSPC detection is less prone to bleachingand movement-induced artefacts compared to wide-field time-gated or frequency-domain FLIM. This microscope is therefore capable of acquiring 3-D FLIM images at significantly increased speeds compared to single beam multiphoton microscopy and we demonstrate this with live cells expressing a GFP tagged protein. We also apply this system to time-lapse FLIM of NAD(P)H autofluorescence in single live cells and report measurements on the change in the fluorescence decay profile following the application of a known metabolic inhibitor.We report a multifocal multiphoton time-correlated single photon counting (TCSPC) fluorescence lifetime imaging (FLIM) microscope system that uses a 16 channel multi-anode PMT detector. Multiphoton excitation minimizes out-of-focus photobleaching, multifocal excitation reduces non-linear in-plane photobleaching effects and TCSPC electronics provide photon-efficient detection of the fluorescence decay profile. TCSPC detection is less prone to bleaching- and movement-induced artefacts compared to wide-field time-gated or frequency-domain FLIM. This microscope is therefore capable of acquiring 3-D FLIM images at significantly increased speeds compared to single beam multiphoton microscopy and we demonstrate this with live cells expressing a GFP tagged protein. We also apply this system to time-lapse FLIM of NAD(P)H autofluorescence in single live cells and report measurements on the change in the fluorescence decay profile following the application of a known metabolic inhibitor.

Toward the clinical application of time-domain fluorescence lifetime imaging

High-speed (video-rate) fluorescence lifetime imaging (FLIM) through a flexible endoscope is reported based on gated optical image intensifier technology. The optimization and potential application of FLIM to tissue autofluorescence for clinical applications are discussed.

Multidimensional Fluorescence Imaging Applied to Biological Tissue

Following the considerable impact of the application of convenient ultrafast lasers to multiphoton microscopy on biomedical imaging, it seems to us that FLIM and MDFI continue the trend in which advances in instrumentation will facilitate new discoveries — and modes of discovery — in biology and medicine. We hope we have shown the reader that fluorescence lifetime can provide intrinsic molecular contrast in unstained tissue and that the prospects for in vivo application are exciting. We believe that the capability to excite fluorophores at almost any excitation wavelength and the opportunities to extract more information from fluorescence signals by resolving with respect to lifetime, excitation and emission spectrum and also polarisation, will have a major impact on the ability to identify and exploit intrinsic contrast and on investigations of molecular biology. There the combination of new fluorescence probe technology, including genetically-expressed labels and nano-engineered devices, with new modes of interrogation and analysis, will continue to fuel the astounding advances in this field. There is a real prospect that our ability to ask and test biological questions will cease to be limited by the availability of suitable instrumentation. Rather it is likely to be limited by our ability to analyse and comprehend the (rapidly increasing volume of) data that we collect.

Spatially selective sampling of single cells using optically trapped fusogenic emulsion droplets: a new single-cell proteomic tool

We present a platform for the spatially selective sampling of the plasma membrane of single cells. Optically trapped lipid-coated oil droplets (smart droplet microtools, SDMs), typically 0.5–5 μm in size, composed of a hexadecane hydrocarbon core and fusogenic lipid outer coating (mixture of 1,2-dioleoyl-phosphatidylethanolamine and 1,2-dioleoyl-sn-glycero-3-phosphatidylcholine) were brought into controlled contact with target colon cancer cells leading to the formation of connecting membrane tethers. Material transfer from the cell to the SDM across the membrane tether was monitored by tracking membrane-localized enhanced green fluorescent protein.

Development of optics with micro-LED arrays for improved opto-electronic neural stimulation

The breakthrough discovery of a nanoscale optically gated ion channel protein, Channelrhodopsin 2 (ChR2), and its combination with a genetically expressed ion pump, Halorhodopsin, allowed the direct stimulation and inhibition of individual action potentials with light alone. This work reports developments of ultra-bright elec tronically controlled optical array sources with enhanced light gated ion channels and pumps for use in systems to further our understanding of both brain and visual function. This work is undertaken as part of the European project, OptoNeuro. Micro-LED arrays permit spatio-temporal control of neuron stimulation on sub-millisecond timescales. However they are disadvantaged by their broad spatial light emission distribution and low fill factor. We present the design and implementation of a projection and micro-optics system for use with a micro-LED array consisting of a 16x16 matrix of 25 μm diameter micro-LEDs with 150 μm centre-to-centre spacing and an emission spectrum centred at 470 nm overlapping the peak sensitivity of ChR2 and its testing on biological samples. The projection system images the micro-LED array onto micro-optics to improve the fill-factor from ~2% to more than 78% by capturing a larger fraction of the LED emission and directing it correctly to the sample plane. This approach allows low fill factor arrays to be used effectively, which in turn has benefits in terms of thermal management and electrical drive from CMOS backplane electronics. The entire projection system is integrated into a microscope prototype to provide stimulation spots at the same size as the neuron cell body (μ10 pm).