Ultrasensitive Label-Free Nanosensing and High-Speed Tracking of Single Proteins

: Label-free detection, analysis, and rapid tracking of nanoparticles is crucial for future ultrasensitive sensing applications, ranging from understanding of biological interactions to the study of size-dependent classical-quantum transitions. Yet optical techniques to distinguish nanoparticles directly among their background remain challenging. Here we present amplified interferometric scattering microscopy (aiSCAT) as a new all-optical method capable of detecting individual nanoparticles as small as 15 kDa proteins that is equivalent to half a GFP. By balancing scattering and reflection amplitudes the interference contrast of the nanoparticle signal is amplified 1 to 2 orders of magnitude. Beyond high sensitivity, a-iSCAT allows high-speed image acquisition exceeding several hundreds of frames-per-second. We showcase the performance of our approach by detecting single Streptavidin binding events and by tracking single Ferritin proteins at 400 frames-per-second with 12 nm localization precision over seconds. Moreover, due to its extremely simple experimental realization, this advancement finally enables a cheap and routine implementation of label-free all-optical single nanoparticle detection platforms with sensitivity operating at the single protein level.Peer ReviewedPostprint (author's final draft

Mr. Newman story

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Calibration choice, rate smoothing, and the pattern of tetrapod diversification according to the long nuclear gene RAG-1

This is an electronic version of an article published in Systematic Biology, 2007; 56 (4):543-563. Systematic Biology is available online at informaworld: http://www.informaworld.com/smpp/content~db=all?content=10.1080/10635150701477825A phylogeny of tetrapods is inferred from nearly complete sequences of the nuclear RAG-1 gene sampled across 88 taxa encompassing all major clades, analyzed via parsimony and Bayesian methods. The phylogeny provides support for Lissamphibia, Theria, Lepidosauria, a turtle-archosaur clade, as well as most traditionally accepted groupings. This tree allows simultaneous molecular clock dating for all tetrapod groups using a set of well-corroborated calibrations. Relaxed clock (PLRS) methods, using the amniote = 315 Mya (million years ago) calibration or a set of consistent calibrations, recovers reasonable divergence dates for most groups. However, the analysis systematically underestimates divergence dates within archosaurs. The bird-crocodile split, robustly documented in the fossil record as being around ∼ 245 Mya, is estimated at only ∼ 190 Mya, and dates for other divergences within archosaurs are similarly underestimated. Archosaurs, and particulary turtles have slow apparent rates possibly confounding rate modeling, and inclusion of calibrations within archosaurs (despite their high deviances) not only improves divergence estimates within archosaurs, but also across other groups. Notably, the monotreme-therian split (∼ 210 Mya) matches the fossil record; the squamate radiation (∼ 190 Mya) is younger than suggested by some recent molecular studies and inconsistent with identification of ∼ 220 and ∼ 165 Myo (million-year-old) fossils as acrodont iguanians and ∼ 95 Myo fossils colubroid snakes; the bird-lizard (reptile) split is considerably older than fossil estimates (≤ 285 Mya); and Sphenodon is a remarkable phylogenetic relic, being the sole survivor of a lineage more than a quarter of a billion years old. Comparison with other molecular clock studies of tetrapod divergences suggests that the common practice of enforcing most calibrations as minima, with a single liberal maximal constraint, will systematically overestimate divergence dates. Similarly, saturation of mitochondrial DNA sequences, and the resultant greater compression of basal branches means that using only external deep calibrations will also lead to inflated age estimates within the focal ingroup.Andrew F. Hugall; Ralph Foster; Michael S. Y. Le

Nucleotide polymorphisms and an improved PCR-based mtDNA diagnostic for parthenogenetic root-knot nematodes (Meloidogyne spp.)

L'analyse de séquences de 2212 paires de bases provenant de six ADN mitochondriaux haplotypes très voisins de #Meloidogyne (Hugall et al., 1994) a permis de mettre en évidence douze sites nucléotidiens polymorphiques et une délétion. En dépit de cette faible diversité, il existe assez de sites différents d'enzymes de restriction parmi ces séquences pour fournir des tests de diagnostic. En utilisant un choix de ces sites, il a été mis au point un test diagnostique multiplex fondé sur l'amplification en chaîne par polymérase (PCR), test amplifiant simultanément deux étroites régions du génome mitochondrial, et digérant ensuite le produit par HinfI ou MnlI. Ce test diagnostique permet d'identifier les haplotypes - même en mélange - présents chez #M. arenaria, #M. incognita, #M. javanica et #M. hispanica, ainsi que chez #M. hapla et #M. chitwoodi. Ce nouveau test représente une amélioration par rapport aux tests PCR sur DNA mitochondrial appliqués aux #Meloidogyne en ce sens qu'il permet la discrimination entre un plus grand nombre d'espèces et de races (par ex. : races de #M. arenaria vis-à-vis de #M. javanica) ; de plus, des produits plus réduits étant amplifiés, ce test est de ce fait plus robuste. Ont été également mis au point des amorces amplifiant une région de 63 paires de bases en nombre variable de répétitions en tandem. Les schémas de zonation qui en résultent permettent de différencier les isolats à l'intérieur des haplotypes d'enzymes de restriction et peuvent être utilisés pour vérifier l'identité des isolats de nématodes maintenus en élevage. (Résumé d'auteur

Nanoscale mapping and control of antenna-coupling strength for bright single photon sources

Cavity QED is the art of enhancing light-matter interaction of photon emitters in cavities, with opportunities for sensing, quantum information and energy capture technologies. To boost emitter-cavity interaction, i.e. coupling strength , ultrahigh quality cavities have been concocted yielding photon trapping times of µs to ms. However, such high-Q cavities give poor photon output, hindering applications. To preserve high photon output it is advantageous to strive for highly localised electric fields in radiatively lossy cavities. Nanophotonic antennas are ideal candidates combining low-Q factors with deeply localised mode volumes, allowing large , provided the emitter is positioned exactly right inside the nanoscale mode volume. Here, with nanometre resolution, we map and tune the coupling strength between a dipole nanoantenna-cavity and a single molecule, obtaining a coupling rate of max ~ 200 GHz. Together with accelerated single photon output, this provides ideal conditions for fast and pure non-classical single photon emission with brightness exceeding 10E9 photons/sec. Clearly, nanoantennas acting as “bad” cavities offer an optimal regime for strong coupling , to deliver bright on-demand and ultrafast single photon nanosources for quantum technologies.Peer ReviewedPostprint (author's final draft

Demonstrating photoluminescence from Au is electronic inelastic light scattering of a plasmonic metal: the origin of SERS backgrounds.

Temperature-dependent surface-enhanced Raman scattering (SERS) is used to investigate the photoluminescence and background continuum always present in SERS but whose origin remains controversial. Both the Stokes and anti-Stokes background is found to be dominated by inelastic light scattering (ILS) from the electrons in the noble metal nanostructures supporting the plasmon modes. The anti-Stokes background is highly temperature dependent and is shown to be related to the thermal occupation of electronic states within the metal via a simple model. This suggests new routes to enhance SERS sensitivities, as well as providing ubiquitous and calibrated real-time temperature measurements of nanostructures.The authors would like to thank EPSRC (EP/K028510/1, EP/ G060649/1, EP/H007024/1, EP/L027151/1), ERC LINASS 320503, EU CUBiHOLE, and Renishaw Diagnostics Ltd. for funding and samples.This is the final published version. It first appeared at http://pubs.acs.org/doi/abs/10.1021/acs.nanolett.5b00146

Plasmonic Nanocavity Coupling

The large losses of plasmonic nanocavities, orders of magnitude beyond those of photonic dielectric cavities, places them, perhaps surprisingly, as exceptional enhancers of single emitter light‐matter interactions. The ultra‐confined, sub‐diffraction limited, mode volumes of plasmonic systems offer huge coupling strengths (in the 1‐100 meV range) to single quantum emitters. Such strengths far outshine the lower coupling strengths of dielectric microcavities, which nonetheless easily achieve single emitter ‘strong coupling’ due to the low loss rates of dielectric cavities. In fact, it is the much higher loss rate of plasmonic cavities that make them desirable for applications requiring bright, fastemitting photon sources. Here we provide a simple method to reformulate lifetime measurements of single emitters in terms of coupling strengths to allow a useful comparison of the literature of plasmonic cavities with that of cavity‐QED, typically more closely associated with dielectric cavities. Using this approach, we observe that the theoretical limit of coupling strength in plasmonic structures has almost been experimentally achieved with even single molecule strong coupling now observed in plasmonic systems. However, key problems remain to maximise the potential of plasmonic cavities, including precise and deterministic nanopositioning of the emitter in the nanosized plasmonic mode volumes, understanding the best geometry for the plasmonic cavity, separating useful photons from background photons and dealing with the fluorescence quenching problems of metals. Here we attempt to raise awareness of the benefits of plasmonic nanocavities for cavity‐QED and tackle some of the potential pitfalls. We observe that there is increasing evidence, that using correct geometries, and improving emitter placement abilities, significant quenching can be avoided and photon output maximised towards the extraordinary limit provided by the high radiative rates of plasmonic nanocavitiesPeer ReviewedPostprint (author's final draft

Phylogeny and biogeography of the camaenid land snails of eastern Australia

Camaenid land snails are the subject of this study, the object of which is the historical biogeography of eastern Australia, with phylogenetic diversity the modus operandi. The thesis is structured around two main sources of information: the distribution data and the phylogenetic data, and makes an empirical contribution to both. Beyond this are inferences on the major evolutionary processes governing biodiversity as seen from this large – or macroecological – perspective. The first chapter introduces three themes: the mosaic of forests along eastern Australia, the camaenid snails and macroecological methods. It also introduces the distribution dataset, issues concerning analysis and provides a picture of the region and the snails’ place in this – the mise en scène. Having stated the case for the need for molecular phylogenetic data, the second chapter describes in detail the gathering and assembly of such data into a comprehensive phylogeny for the snails. In doing so it makes a substantial contribution to the higher taxonomy of the whole group, and to the methodology of supermatrix ‘tree-of-life’ phylogenetics. The results suggest that the camaenids stem from Oligo-Miocene Laurasian immigration. Relictual endemics indicate that many ancestral lineages were in place before the major decline of the mesic forest realm. Polyphyly of many genera highlight the repeated radiation of shell forms and that the higher taxonomy was unacceptable. The results provide the basis of a new generic framework. The subsequent chapters use the resources now provided to investigate historical and environmental influences on biodiversity at different phylogenetic and spatial scales: within a single species in one region; a clade (genus) spread across regions; the entire biota. Chapter 3 investigates in detail a single species in a single well studied region, the Wet Tropics rainforests. This multifaceted approach, combining spatially explicit paleoclimatological models and comparative phylogeography, provides a powerful approach to locating historical refugia, highlighting the role of landscape, ecology and history in shaping population structure and hence the foundations of allopatric speciation. The next chapter expands this to encompass the whole clade – the Sphaerospira lineage – spanning the ‘mesotherm archipelago’ of mesic forest along eastern Australia. A trans-species phylogeny is combined with bioclimatic modelling, spatial mapping of phylogenetic diversity and lineage diversification analyses to reveal the profound link between the intra-specific phylogeography and the underlying inter-specific phylogeny. The final chapter (5) represents a culmination of the ideas and data introduced in the previous sections to: i) explore methods of incorporating phylogenetic information into biogeography and macroecology; ii) provide a detailed and comprehensive biogeography of eastern Australia; iii) speculate upon major patterns and processes in biodiversity: speciation, accumulation, retention, extinction. However, it does come to some broad conclusions: that diversification proceeds from peripheral isolation, is driven by environmental gradients; that diversity is governed by environment through extinction, with ecosystem turnover due to the late Tertiary aridification.Thesis (Ph.D.) -- University of Adelaide, School of Biological Sciences, 201

On the nature of SERS from plasmonic nanostructures

The nature of surface-enhanced Raman scattering (SERS) on nanostructured surfaces is explored using both inorganic and organic-based systems and a variety of environmental perturbation mechanisms. Experimental optical characterisation systems are developed and existing systems extended to facilitate this exploration. SERS of inorganic semiconducting quantum dots (QDs) is observed for the first time, paving the way for their use as spatially well-defined SERS markers. Tuning of the Raman excitation wavelength allows comparison between resonance and nonresonance QD SERS and identifies enhancement due to the plasmonic nanostructure. A gentle mechano-chemical process (carbon dioxide snow jet) is used to rearrange adsorbed organic thiol monolayers on a gold plasmonic nanostructure. The necessity of nanoscale roughness to the large SERS enhancement on pit-like plasmonic nanostructures is shown and demonstrates a new method to boost SERS signals (> 500 %) on plasmonic nanostructures. A multiplexed time-varied exposure technique is developed to track this molecular movement over time and highlights the different origins of the SERS peak and its accompanying background continuum. Using low-temperature cryogenics (down to 10 K) the SERS peak and background continuum intensity are shown to increase as the plasmonic metal damping reduces with temperature. Temperature dependent measurements of QD (resonance) SERS are shown to have strong wavelength dependence due to the excitonic transitions in QDs. Changes to the QD fluorescence at low temperature allows striking comparison between the Raman and fluorescence processes. The role of charge transfer and electromagnetic enhancement in the SERS intensity of p-aminothiophenol (pATP) is investigated on nanostructured plasmonic surfaces coupled to metallic nanoparticles. The results support the importance of charge transfer effects to the SERS of pATP, and highlight the difference between those of electromagnetic origin. Addition of nanoparticles to the nanostructured surface was seen to enhance SERS signals by up to 100×.Funded by an EPSRC Industrial CASE studentship in collaboration with Renishaw Diagnostics Ltd

Two distinct forms of Chlamydia psittaci associated with disease and infertility in Phascolarctos cinereus (Koala)

While several diseases associated with Chlamydia psittaci infection have been reported in Phascolarctos cinereus (koala), it is still unclear whether one or more chlamydial strains are responsible. In this study, we provide evidence, obtained by restriction enzyme and gene probe analysis, that two quite distinct strains of C. psittaci infect koalas; one strain was isolated from the conjunctivae, and the other was isolated from the urogenital tract and the rectum. A gene probe, pFEN207, containing the coding sequence for an enzyme involved in the biosynthesis of the chlamydial genus-specific lipopolysaccharide antigen, and a separate probe, pCPML-4N, prepared from a DNA fragment of a koala-infecting strain of C. psittaci, were used to determine the patterns of hybridization in the koala-infecting strains; these patterns were found to be quite distinct from those observed with C. psittaci isolates from other animals. We also demonstrated by hybridization analysis with an avian strain plasmid that all three koala urogenital isolates contain a plasmid and that there is no evidence for the presence of a homologous plasmid in any of the ocular isolates