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ABSTRACT: Localization based super-resolution microscopy techniques require precise drift correction methods because the achieved spatial resolution is close to both the mechanical and optical performance limits of modern light microscopes. Multi-color imaging methods require corrections in addition to those dealing with drift due to the static, but spatially-dependent, chromatic offset between images. We present computer simulations to quantify this effect, which is primarily caused by the high-NA objectives used in super-resolution microscopy. Although the chromatic offset in well corrected systems is only a fraction of an optical wavelength in magnitude (<50 nm) and thus negligible in traditional diffraction limited imaging, we show that object colocalization by multi-color super-resolution methods is impossible without appropriate image correction. The simulated data are in excellent agreement with experiments using fluorescent beads excited and localized at multiple wavelengths. Finally we present a rigorous and practical calibration protocol to correct for chromatic optical offset, and demonstrate its efficacy for the imaging of transferrin receptor protein colocalization in HeLa cells using two-color direct stochastic optical reconstruction microscopy (dSTORM).
Optics Express 05/2013; 21(9):10978-88. · 3.59 Impact Factor
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ABSTRACT: Kinetic assay of seeded growth: The graph shows the variation in intrinsic fluorescence intensity of amyloid fibrils. Fluorescence increases during the seeded aggregation of α-synuclein seeds with α-synuclein monomeric protein (blue curve) but not when α-synuclein seeds are incubated with β-synuclein monomeric protein (black curve), thus showing that no seeded growth occurred in this case.
ChemBioChem 04/2013; · 3.94 Impact Factor
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ABSTRACT: We report observations of an intrinsic fluorescence in the visible range, which develops during the aggregation of a range of polypeptides, including the disease-related human peptides amyloid-β(1-40) and (1-42), lysozyme and tau. Characteristic fluorescence properties such as the emission lifetime and spectra were determined experimentally. This intrinsic fluorescence is independent of the presence of aromatic side-chain residues within the polypeptide structure. Rather, it appears to result from electronic levels that become available when the polypeptide chain folds into a cross-β sheet scaffold similar to what has been reported to take place in crystals. We use these findings to quantify protein aggregation in vitro by fluorescence imaging in a label-free manner.
The Analyst 04/2013; 138(7):2156-62. · 4.23 Impact Factor
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ABSTRACT: We present a numerical study of interactions between dispersive waves (DWs) and solitons during supercontinuum generation in photonic crystal fibers pumped with picosecond laser pulses. We show how the soliton-induced trapping potential evolves along the fiber and affects the dynamics of a DW-soliton pair. Individual frequency components of the DW periodically interact with the soliton resulting in stepwise frequency blue shifts. In contrast, the ensemble blue shifts of all frequency components in the DW appear to be quasi-continuous. The step size of frequency up-conversion and the temporal separation between subsequent soliton-DW interactions are governed by the potential well which confines the soliton-DW pair and which changes in time.
Optics Express 03/2012; 20(6):6316-24. · 3.59 Impact Factor
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ABSTRACT: Fibril formation by mutational variants of human lysozyme is associated with a fatal form of hereditary non-neuropathic systemic amyloidosis. Defining the mechanistic details of lysozyme aggregation is of crucial importance for understanding the origin and progression of this disease and related misfolding conditions. In this study, we show that a biotin moiety can be introduced site-specifically at Lys33 of human lysozyme. We demonstrate, using biophysical techniques, that the structure and stability of the native-state of the protein are not detectably altered by this modification, and that the ability to form amyloid fibrils is unchanged. By taking advantage of biotin-avidin interactions, we show that super-resolution fluorescence microscopy can generate detailed images of the mature fibrils. This methodology can readily enable the introduction of additional probes into the protein, thereby providing the means through which to understand, in detail, the nature of the aggregation process of lysozyme and its variants under a variety of conditions.
PLoS ONE 01/2012; 7(11):e50192. · 4.09 Impact Factor
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Tetsuro Murakami,
Seung-Pil Yang,
Lin Xie,
Taizo Kawano,
Donald Fu,
Asuka Mukai,
Christopher Bohm,
Fusheng Chen,
Janice Robertson,
Hiroshi Suzuki,
Gian Gaetano Tartaglia,
Michele Vendruscolo,
Gabriele S Kaminski Schierle,
Fiona T S Chan,
Aileen Moloney,
Damian Crowther, Clemens F Kaminski,
Mei Zhen,
Peter St George-Hyslop
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ABSTRACT: It is unclear whether mutations in fused in sarcoma (FUS) cause familial amyotrophic lateral sclerosis via a loss-of-function effect due to titrating FUS from the nucleus or a gain-of-function effect from cytoplasmic overabundance. To investigate this question, we generated a series of independent Caenorhabditis elegans lines expressing mutant or wild-type (WT) human FUS. We show that mutant FUS, but not WT-FUS, causes cytoplasmic mislocalization associated with progressive motor dysfunction and reduced lifespan. The severity of the mutant phenotype in C. elegans was directly correlated with the severity of the illness caused by the same mutation in humans, arguing that this model closely replicates key features of the human illness. Importantly, the mutant phenotype could not be rescued by overexpression of WT-FUS, even though WT-FUS had physiological intracellular localization, and was not recruited to the cytoplasmic mutant FUS aggregates. Our data suggest that FUS mutants cause neuronal dysfunction by a dominant gain-of-function effect related either to neurotoxic aggregates of mutant FUS in the cytoplasm or to dysfunction in its RNA-binding functions.
Human Molecular Genetics 09/2011; 21(1):1-9. · 7.64 Impact Factor
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ABSTRACT: The broad family of LEA proteins are intrinsically disordered proteins (IDPs) with several potential roles in desiccation tolerance, or anhydrobiosis, one of which is to limit desiccation-induced aggregation of cellular proteins. We show here that this activity, termed molecular shield function, is distinct from that of a classical molecular chaperone, such as HSP70 - while HSP70 reduces aggregation of citrate synthase (CS) on heating, two LEA proteins, a nematode group 3 protein, AavLEA1, and a plant group 1 protein, Em, do not; conversely, the LEA proteins reduce CS aggregation on desiccation, while HSP70 lacks this ability. There are also differences in interaction with client proteins - HSP70 can be co-immunoprecipitated with a polyglutamine-containing client, consistent with tight complex formation, whereas the LEA proteins can not, although a loose interaction is observed by Förster resonance energy transfer. In a further exploration of molecular shield function, we demonstrate that synthetic polysaccharides, like LEA proteins, are able to reduce desiccation-induced aggregation of a water-soluble proteome, consistent with a steric interference model of anti-aggregation activity. If molecular shields operate by reducing intermolecular cohesion rates, they should not protect against intramolecular protein damage. This was tested using the monomeric red fluorescent protein, mCherry, which does not undergo aggregation on drying, but the absorbance and emission spectra of its intrinsic fluorophore are dramatically reduced, indicative of intramolecular conformational changes. As expected, these changes are not prevented by AavLEA1, except for a slight protection at high molar ratios, and an AavLEA1-mCherry fusion protein is damaged to the same extent as mCherry alone. A recent hypothesis proposed that proteomes from desiccation-tolerant species contain a higher degree of disorder than intolerant examples, and that this might provide greater intrinsic stability, but a bioinformatics survey does not support this, since there are no significant differences in the degree of disorder between desiccation tolerant and intolerant species. It seems clear therefore that molecular shield function is largely an intermolecular activity implemented by specialist IDPs, distinct from molecular chaperones, but with a role in proteostasis.
Molecular BioSystems 09/2011; 8(1):210-9. · 3.53 Impact Factor
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ABSTRACT: Misfolding and aggregation of peptides and proteins is a characteristic of many neurodegenerative disorders, including Alzheimer's disease (AD). In AD the β-amyloid peptide (Aβ) aggregates to form characteristic fibrillar structures, which are the deposits found as plaques in the brains of patients. We have used direct stochastic optical reconstruction microscopy, dSTORM, to probe the process of in situ Aβ aggregation and the morphology of the ensuing aggregates with a resolution better than 20 nm. We are able to distinguish different types of structures, including oligomeric assemblies and mature fibrils, and observe a number of morphological differences between the species formed in vitro and in vivo, which may be significant in the context of disease. Our data support the recent view that intracellular Aβ could be associated with Aβ pathogenicity in AD, although the major deposits are extracellular, and suggest that this approach will be widely applicable to studies of the molecular mechanisms of protein deposition diseases.
Journal of the American Chemical Society 08/2011; 133(33):12902-5. · 9.91 Impact Factor
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ABSTRACT: The anesthetic agent propofol (2,6-diisopropylphenol) is the most widely used intravenously administered drug in general anesthesia. However, a viable online capability to monitor metabolized levels of propofol in patients does not currently exist. Here we show for the first time that optical spectroscopy has good potential to detect metabolized propofol from patients' exhaled breath. We present quantitative absorption measurements of gas phase propofol both in the ultraviolet and middle-infrared spectral regions. We demonstrate that a detection limit in the subparts-per-billion concentration range can be reached with photoacoustic spectroscopy in the UV spectral region, paving the way for the development of future optical monitors.
Analytical Chemistry 05/2011; 83(10):3963-7. · 5.86 Impact Factor
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ABSTRACT: Plasmodium falciparum is responsible for severe malaria. During the ∼48 h duration of its asexual reproduction cycle in human red blood cells, the parasite causes profound alterations in the homeostasis of the host red cell, with reversal of the normal Na and K gradients across the host cell membrane, and a drastic fall in hemoglobin content. A question critical to our understanding of how the host cell retains its integrity for the duration of the cycle had been previously addressed by modeling the homeostasis of infected cells. The model predicted a critical contribution of excess hemoglobin consumption to cell integrity (the colloidosmotic hypothesis). Here we tested this prediction with the use of electron-probe x-ray microanalysis to measure the stage-related changes in Na, K, and Fe contents in single infected red cells and in uninfected controls. The results document a decrease in Fe signal with increased Na/K ratio. Interpreted in terms of concentrations, the results point to a sustained fall in host cell hemoglobin concentration with parasite maturation, supporting a colloidosmotic role of excess hemoglobin digestion. The results also provide, for the first time to our knowledge, comprehensive maps of the elemental distributions of Na, K, and Fe in falciparum-infected red blood cells.
Biophysical Journal 03/2011; 100(6):1438-45. · 3.65 Impact Factor
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ChemPhysChem 02/2011; 12(3):423-4. · 3.41 Impact Factor
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Gabriele S Kaminski Schierle,
Carlos W Bertoncini,
Fiona T S Chan,
Annemieke T van der Goot,
Stefanie Schwedler,
Jeremy Skepper,
Simon Schlachter,
Tjakko van Ham,
Alessandro Esposito,
Janet R Kumita,
Ellen A A Nollen,
Christopher M Dobson, Clemens F Kaminski
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ABSTRACT: Misfolding and aggregation of amyloidogenic polypeptides lie at the root of many neurodegenerative diseases. Whilst protein aggregation can be readily studied in vitro by established biophysical techniques, direct observation of the nature and kinetics of aggregation processes taking place in vivo is much more challenging. We describe here, however, a Förster resonance energy transfer sensor that permits the aggregation kinetics of amyloidogenic proteins to be quantified in living systems by exploiting our observation that amyloid assemblies can act as energy acceptors for variants of fluorescent proteins. The observed lifetime reduction can be attributed to fluorescence energy transfer to intrinsic energy states associated with the growing amyloid species. Indeed, for a-synuclein, a protein whose aggregation is linked to Parkinson's disease, we have used this sensor to follow the kinetics of the self-association reactions taking place in vitro and in vivo and to reveal the nature of the ensuing aggregated species. Experiments were conducted in vitro, in cells in culture and in living Caenorhabditis elegans. For the latter the readout correlates directly with the appearance of a toxic phenotype. The ability to measure the appearance and development of pathogenic amyloid species in a living animal and the ability to relate such data to similar processes observed in vitro provides a powerful new tool in the study of the pathology of the family of misfolding disorders. Our study confirms the importance of the molecular environment in which aggregation reactions take place, highlighting similarities as well as differences between the processes occurring in vitro and in vivo, and their significance for defining the molecular physiology of the diseases with which they are associated.
ChemPhysChem 02/2011; 12(3):673-80. · 3.41 Impact Factor
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ABSTRACT: Molecular self-assembly is a defining feature of numerous biological functions and dysfunctions, ranging from basic cell signalling to diseases mediated by protein aggregation. There is current demand for novel experimental methods to study molecular self-assembly in live cells, and thereby in its physiological context. Förster resonance energy transfer (FRET) between fluorophores of a single type, known as homoFRET, permits noninvasive detection and quantification of molecular clusters in live cells. It can thus provide powerful insights into the molecular physiology of living systems and disease. HomoFRET is detected by measuring the loss of fluorescence anisotropy upon excitation with polarised light. This article reviews recent key developments in homoFRET fluorescence anisotropy imaging for the detection and quantification of molecular self-assembly reactions in biological systems. A summary is given of the current state-of-the-art and case studies are presented of successful implementations, highlighting technical aspects which have to be mastered to bridge the gap between proof-of-concept experiments and biological discoveries.
ChemPhysChem 02/2011; 12(3):500-9. · 3.41 Impact Factor
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ABSTRACT: Biophysical imaging tools exploit several properties of fluorescence to map cellular biochemistry. However, the engineering of a cost-effective and user-friendly detection system for sensing the diverse properties of fluorescence is a difficult challenge. Here, we present a novel architecture for a spectrograph that permits integrated characterization of excitation, emission and fluorescence anisotropy spectra in a quantitative and efficient manner. This sensing platform achieves excellent versatility of use at comparatively low costs. We demonstrate the novel optical design with example images of plant cells and of mammalian cells expressing fluorescent proteins undergoing energy transfer.
Optics Express 01/2011; 19(3):2546-55. · 3.59 Impact Factor
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ABSTRACT: We present an adaptive numerical filter for analyzing fiber-length dependent properties of optical rogue waves, which are highly intense and extremely red-shifted solitons that arise during supercontinuum generation in photonic crystal fiber. We use this filter to study a data set of 1000 simulated supercontinuum pulses, produced from 5 ps pump pulses containing random noise. Optical rogue waves arise in different supercontinuum pulses at various positions along the fiber, and exhibit a lifecycle: their intensity peaks over a finite range of fiber length before declining slowly.
Optics Express 12/2010; 18(25):26113-22. · 3.59 Impact Factor
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ABSTRACT: Interactions between supercontinuum (SC) light pulses, produced by the propagation of rapidly sequenced picosecond pump laser pulses along a photonic crystal fiber, result in spectral broadening, which we attribute to interpulse soliton collisions. This phenomenon was measured experimentally, following our observation of spectral broadening in numerical simulations that exhibit so-called "pulse wraparound" or "temporal aliasing." This occurs in simulations with narrow time grids: as early parts of the SC pulse leave the computational time domain, they "reenter" at the beginning and so interact with later parts of the evolving SC pulse. We show that this provides an effective model to predict the experimentally observed spectral changes.
Optics Letters 12/2010; 35(24):4145-7. · 3.40 Impact Factor
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ABSTRACT: Investigation of the homeostasis of red blood cells upon infection by Plasmodium falciparum poses complex experimental challenges. Changes in red cell shape, volume, protein, and ion balance are difficult to quantify. In this article, we review a wide range of optical techniques for quantitative measurements of critical homeostatic parameters in malaria-infected red blood cells. Fluorescence lifetime imaging and tomographic phase microscopy, quantitative deconvolution microscopy, and X-ray microanalysis, are used to measure haemoglobin concentration, cell volume, and ion contents. Atomic force microscopy is briefly reviewed in the context of these optical methodologies. We also describe how optical tweezers and optical stretchers can be usefully applied to empower basic malaria research to yield diagnostic information on cell compliance changes upon malaria infection. The combined application of these techniques sheds new light on the detailed mechanisms of malaria infection providing potential for new diagnostic or therapeutic approaches.
Medical & Biological Engineering 10/2010; 48(10):1055-63. · 1.76 Impact Factor
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ABSTRACT: Intrinsically disordered proteins (IDPs) lack well-defined structure but are widely represented in eukaryotic proteomes. Although the functions of most IDPs are not understood, some have been shown to have molecular recognition and/or regulatory roles where their disordered nature might be advantageous. Anhydrin is an uncharacterized IDP induced by dehydration in an anhydrobiotic nematode, Aphelenchus avenae. We show here that anhydrin is a moonlighting protein with two novel, independent functions relating to desiccation tolerance. First, it has a chaperone-like activity that can reduce desiccation-induced enzyme aggregation and inactivation in vitro. When expressed in a human cell line, anhydrin localizes to the nucleus and reduces the propensity of a polyalanine expansion protein associated with oculopharyngeal muscular dystrophy to form aggregates. This in vivo activity is distinguished by a loose association of anhydrin with its client protein, consistent with a role as a molecular shield. In addition, anhydrin exhibits a second function as an endonuclease whose substrates include supercoiled, linear, and chromatin linker DNA. This nuclease activity could be involved in either repair of desiccation-induced DNA damage incurred during anhydrobiosis or in apoptotic or necrotic processes, for example, but it is particularly unexpected for anhydrin because IDP functions defined to date anticorrelate with enzyme activity. Enzymes usually require precise three-dimensional positioning of residues at the active site, but our results suggest this need not be the case. Anhydrin therefore extends the range of IDP functional categories to include catalysis and highlights the potential for the discovery of new functions in disordered proteomes.
Proceedings of the National Academy of Sciences 09/2010; 107(37):16084-9. · 9.68 Impact Factor
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ABSTRACT: We demonstrate a step change in the capability of diode laser excited two-line atomic fluorescence (TLAF) thermometry and show it is well-suited to the study of low-pressure sooting flames. The new devel-opments to the technique reported here were essential to achieve the accuracy (±41 K) and precision (±8 K) required for useful measurements in such systems. This represents the first application of TLAF thermometry to the investigation of low-pressure sooting flames, an environment in which other thermom-etry techniques perform poorly. We thus demonstrate a practical application of diode laser TLAF to a bur-ner that is the subject of a coordinated experimental and computational investigation of soot formation. The TLAF technique requires no calibration measurement and is compact and economical to set up in comparison with traditional laser thermometry methods. Temperature profiles were recorded in a laminar flat-flame operating on O 2 , N 2 and CH 4 at fuel equivalence ratio of 2.32 and pressures ranging from 18.7 to 26.7 kPa. Almost identical temperature profiles were observed at different pressures despite the fact that soot volume fractions changed by more than an order of magnitude between the lowest and highest oper-ating pressures. The data will contribute to modelling efforts to understand the surprisingly strong depen-dence of soot volume fraction on pressure that has previously been observed under the range of conditions studied here and will be included in an openly available database on this flame, which includes species pro-file measurements obtained by other methods. In the current contribution we emphasise the technical implementation of diode TLAF as a new temperature diagnostic with near optimal characteristics for the study of low pressure, sooting flames.
09/2010;
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ABSTRACT: A novel spectrometer for the rapid and sensitive detection of liquid phase analytes at trace concentrations is presented. Broad bandwidth supercontinuum radiation was coupled into a linear optical cavity incorporating an intracavity liquid-sample cuvette. Cavity enhanced absorption spectra of trace species covering more than 300 nm were acquired on time scales of milliseconds. Single shot acquisition times of 10-50 ms are demonstrated here. The effective absorption path length exceeds 2 m in sample volumes measuring 2.7 mL. A key feature of the instrument is that it can be calibrated using cavity ring-down spectroscopy without the requirement of changing the optical alignment. The sensitivity of the instrument is exemplified by measurements of trace concentrations of dye molecules and nickel sulfate dissolved in water. A minimum detectable absorption coefficient of 9.1 x 10(-7) cm(-1) Hz(-1/2) at 550 nm was obtained. The capability to capture broad bandwidth absorption spectra on short time scales permits kinetic studies of liquid phase reactions. We demonstrate this by recording the oscillatory behavior of a Belousov-Zhabotinsky reaction.
Analytical Chemistry 09/2010; 82(17):7498-501. · 5.86 Impact Factor
