Multidimensional Fluorescence Imaging and Super-resolution Exploiting Ultrafast Laser and Supercontinuum Technology

This thesis centres on the development of multidimensional fluorescence imaging tools, with a particular emphasis on fluorescence lifetime imaging (FLIM) microscopy for application to biological research. The key aspects of this thesis are the development and application of tunable supercontinuum excitation sources based on supercontinuum generation in microstructured optical fibres and the development of stimulated emission depletion (STED) microscope capable of fluorescence lifetime imaging beyond the diffraction limit. The utility of FLIM for biological research is illustrated by examples of experimental studies of the molecular structure of sarcomeres in muscle fibres and of signalling at the immune synapse. The application of microstructured optical fibre to provide tunable supercontinuum excitation source for a range of FLIM microscopes is presented, including wide-field, Nipkow disk confocal and hyper-spectral line scanning FLIM microscopes. For the latter, a detailed description is provided of the supercontinuum source and semi-confocal line-scanning microscope configuration that realised multidimensional fluorescence imaging, resolving fluorescence images with respect to excitation and emission wavelength, fluorescence lifetime and three spatial dimensions. This included the first biological application of a fibre laser-pumped supercontinuum exploiting a tapered microstructured optical fibre that was able to generate a spectrally broad output extending to ~ 350 nm in the ultraviolet. The application of supercontinuum generation to the first super-resolved FLIM microscope is then described. This novel microscope exploited the concept of STED with a femtosecond mode-locked Ti:Sapphire laser providing a tunable excitation beam by pumping microstructured optical fibre for supercontinuum generation and directly providing the (longer wavelength) STED beam. This STED microscope was implemented in a commercial scanning confocal microscope to provide compatibility with standard biological imaging, and exploited digital holography using a spatial light modulator (SLM) to provide the appropriate phase manipulation for shaping the STED beam profile and to compensate for aberrations. The STED microscope was shown to be capable of recording super resolution in both the lateral and axial planes, according to the settings of the SLM

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

Compact and Mobile Full-Field Optical Coherence Tomography Sensor for Subsurface Fingerprint Imaging

Conventional fingerprint sensors that are deployed in real-life applications lack the ability to peer inside a finger beyond the external surface. Subsurface information can provide complimentary biometric characteristics associated with the finger. The subsurface fingerprints can also be employed when the quality of the external/surface fingerprints is affected. One of the most promising technologies for imaging below the surface of an external fingerprint is full-field optical coherent tomography (FF-OCT). However, the FF-OCT can be expensive and cumbersome, despite its proven ability for biometric use. In this paper, we describe the design and implementation of a compact, mobile and cost-effective FF-OCT sensor that is stable and easy to use. The newly designed sensor, being 30 cm × 30 cm × 10 cm in size, comprises of a dedicated silicon camera, stable Michelson interferometer and a bright Near-Infra-Red (NIR) light emitting diode. It enables recording of 1.7 cm × 1.7 cm images of subsurface finger features, such as internal fingerprints and sweat ducts. We show the employability of the newly designed sensor for different applications. Specifically, we validate its usefulness by capturing subsurface fingerprints of 585 subjects leading to 3510 unique fingerprints. The resulting accuracy of 0.74% as Equal Error Rate (EER) indicates the backward compatibility of the proposed sensor with the existing commercial off-the-shelf algorithms. Thanks to the large fingerprint database collected in this work we determined the most useful imaging depth for the fingerprint matching purposes to be around 100 gym. As an additional advantage, the sensor could be readily used in other applications with little or no modification, such as in vivo skin imaging

Functionalized luminescent oxide nanoparticles for sodium channel imaging at the single molecule level

International audienceLanthanide-ion doped oxide nanoparticles were functionalized for use as fluorescent biological labels. These nanoparticles are synthesized directly in water which facilitates their functionalization, and are very photostable without emission intermittency. Nanoparticles functionalized with guanidinium groups act as artificial toxins and specifically target sodium channels. They are individually detectable in cardiac myocytes, revealing a heterogeneous distribution of sodium channels. Functionalized oxide nanoparticles appear as a novel tool particularly well adapted to long-term single-molecule tracking. © (2005) COPYRIGHT SPIE--The International Society for Optical Engineerin

Media 2: Dual-modality fluorescence and full-field optical coherence microscopy for biomedical imaging applications

Originally published in Biomedical Optics Express on 01 March 2012 (boe-3-3-661