In JoVE (1)

Other Publications (64)

Articles by Clemens F. Kaminski in JoVE

Other articles by Clemens F. Kaminski on PubMed

Maximum-likelihood Curve-fitting Scheme for Experiments with Pulsed Lasers Subject to Intensity Fluctuations

Applied Optics. Mar, 2003  |  Pubmed ID: 12665086

Evaluation schemes, e.g., least-squares fitting, are not generally applicable to any types of experiments. If the evaluation schemes were not derived from a measurement model that properly described the experiment to be evaluated, poorer precision or accuracy than attainable from the measured data could result. We outline ways in which statistical data evaluation schemes should be derived for all types of experiment, and we demonstrate them for laser-spectroscopic experiments, in which pulse-to-pulse fluctuations of the laser power cause correlated variations of laser intensity and generated signal intensity. The method of maximum likelihood is demonstrated in the derivation of an appropriate fitting scheme for this type of experiment. Statistical data evaluation contains the following steps. First, one has to provide a measurement model that considers statistical variation of all enclosed variables. Second, an evaluation scheme applicable to this particular model has to be derived or provided. Third, the scheme has to be characterized in terms of accuracy and precision. A criterion for accepting an evaluation scheme is that it have accuracy and precision as close as possible to the theoretical limit. The fitting scheme derived for experiments with pulsed lasers is compared to well-established schemes in terms of fitting power and rational functions. The precision is found to be as much as three timesbetter than for simple least-squares fitting. Our scheme also suppresses the bias on the estimated model parameters that other methods may exhibit if they are applied in an uncritical fashion. We focus on experiments in nonlinear spectroscopy, but the fitting scheme derived is applicable in many scientific disciplines.

Wide-bandwidth Mode-hop-free Tuning of Extended-cavity GaN Diode Lasers

Applied Optics. Jun, 2005  |  Pubmed ID: 15989042

We present a new approach for extended-cavity diode-laser tuning to achieve wide mode-hop-free tuning ranges. By using a multiple piezoactuated grating mount, the cavity length and grating angle in the laser can be adjusted independently, allowing mode-hop-free tuning without the need for a mechanically optimized pivot-point mount. Furthermore, synchronized diode injection-current tuning allows diode lasers without antireflection coatings to be employed. In combination these two techniques make the construction of a cheap, efficient, and easily optimized extended-cavity diode laser possible. A theoretical analysis is presented for optimal control of piezoactuator displacements and injection current to achieve the widest possible mode-hop-free tuning ranges, and a comparison is made with measurements. The scheme is demonstrated for blue and violet GaN lasers operating at approximately 450 nm and approximately 410 nm, for which continuous tuning ranges exceeding 90 GHz have been achieved. Examples of applications of these lasers are also given.

Quantitative Kinetic Analysis in a Microfluidic Device Using Frequency-domain Fluorescence Lifetime Imaging

Analytical Chemistry. Jun, 2007  |  Pubmed ID: 17472341

A novel microfluidic approach for the quantification of reaction kinetics is presented. A three-dimensional finite difference numerical simulation was developed in order to extract quantitative kinetic information from fluorescence lifetime imaging experimental data. This approach was first utilized for the study of a fluorescence quenching reaction within a microchannel; the lifetime of a fluorophore was used to map the diffusion of a quencher across the microchannel. The approach was then applied to a more complex chemical reaction between a fluorescent amine and an acid chloride, via numerical simulation the bimolecular rate constant for this reaction was obtained.

High Bandwidth Absorption Spectroscopy with a Dispersed Supercontinuum Source

Optics Express. Sep, 2007  |  Pubmed ID: 19547496

An optical gas sensor is presented, making use of a dispersed supercontinuum source, capable of acquiring broad bandwidth spectra at ultrahigh wavelength sweep and repetition rates. Wavelength sweeps from 1100 nm to 1700 nm can be performed in 800 ns at a spectral resolution of 40 pm. This is comparable to line-widths of molecular spectra at atmospheric pressure. Quantitative measurements are presented of CH(4) employing 80 nm wide sweeps over the P- Q- and R-branches of the 2nu(3) transition near 1665 nm, at rates exceeding 100 kHz. The effective acquisition rate is determined by the amount of averaging required, and the effect of this averaging on observed precision is investigated.

Theoretical Investigation of the Photon Efficiency in Frequency-domain Fluorescence Lifetime Imaging Microscopy

Journal of the Optical Society of America. A, Optics, Image Science, and Vision. Feb, 2008  |  Pubmed ID: 18246179

We investigate the photon efficiency of frequency-domain fluorescence lifetime imaging microscopy, using both theoretical and Monte Carlo methods. Our analysis differs from previous work in that it incorporates the data fitting process used in real experiments, allows for the arbitrary choice of excitation and gain waveforms, and calculates lifetimes as well as associated F-values from higher harmonics in the data. Using our analysis, we found different photon efficiencies to those previously reported and were able to propose optimal excitation and gain waveforms. Additionally, we suggest measurement protocols that lead to further improvement in photon efficiency. We compare our results to other techniques for lifetime imaging and consider the implications of our higher-harmonic analysis for multi-exponential lifetime determination.

Fluorescence Intensity and Lifetime Imaging of Free and Micellar-encapsulated Doxorubicin in Living Cells

Nanomedicine : Nanotechnology, Biology, and Medicine. Mar, 2008  |  Pubmed ID: 18249155

Frequency domain fluorescence lifetime imaging microscopy (FLIM) has been used in combination with laser scanning confocal microscopy to study the cellular uptake behavior of the antitumor drug doxorubicin (DOX) and micellar-encapsulated DOX (PLyAd-DOX). The endocytosis uptake process of PLyAd-DOX was monitored over 72 hours using confocal microscopy, with a maximum fluorescence recorded at incubation periods around 24 hours. The micellar structure was not found to release the encapsulated DOX during the time course of imaging. FLIM revealed single lifetime distributions of PLyAd-DOX during accumulation in the cytoplasm. The free DOX in contrast was observed both in the cytoplasm and the nuclear domain of the cell, showing bimodal lifetime distributions. There was a marked dependence of the measured free-DOX lifetime on concentration within the cell, in contrast to reference experiments in aqueous solution, where no such dependence was found. The results suggest the formation of macromolecular structures inside the living cells.

From Microdroplets to Microfluidics: Selective Emulsion Separation in Microfluidic Devices

Angewandte Chemie (International Ed. in English). 2008  |  Pubmed ID: 18264960

Direct Visualization of Reversible Switching of Micropatterned Polyelectrolyte Brushes on Gold Surfaces Using Laser Scanning Confocal Microscopy

Langmuir : the ACS Journal of Surfaces and Colloids. Nov, 2008  |  Pubmed ID: 18842063

We apply confocal fluorescence microscopy for real time studies of reversible conformational changes of poly(methacryloyloxyethyl phosphate) (PMEP) brushes chemically grafted onto gold substrates. Oregon green 488 fluorophores chemically attached onto the PMEP polymers were used as reporters for probing the conformational changes. Use of a specially designed liquid flow microchamber allowed dynamic imaging of the brushes under varying environmental conditions. The fluorescence intensities exhibited fully reversible brightness changes on alternation of the solution in the chamber between water and KCl. This reversible quenching behavior is consistent with a conformational change between an extended and a collapsed brush configuration. The fluorescence quenching behavior of the brushes was found to be dependent on ion concentration as well as polymer grafting density and was caused by nonradiative energy transfer to the polymer scaffold and the gold substrate.

FRET Imaging of Hemoglobin Concentration in Plasmodium Falciparum-infected Red Cells

PloS One. 2008  |  Pubmed ID: 19023444

During its intraerythrocytic asexual reproduction cycle Plasmodium falciparum consumes up to 80% of the host cell hemoglobin, in large excess over its metabolic needs. A model of the homeostasis of falciparum-infected red blood cells suggested an explanation based on the need to reduce the colloid-osmotic pressure within the host cell to prevent its premature lysis. Critical for this hypothesis was that the hemoglobin concentration within the host cell be progressively reduced from the trophozoite stage onwards.

The Homeostasis of Plasmodium Falciparum-infected Red Blood Cells

PLoS Computational Biology. Apr, 2009  |  Pubmed ID: 19343220

The asexual reproduction cycle of Plasmodium falciparum, the parasite responsible for severe malaria, occurs within red blood cells. A merozoite invades a red cell in the circulation, develops and multiplies, and after about 48 hours ruptures the host cell, releasing 15-32 merozoites ready to invade new red blood cells. During this cycle, the parasite increases the host cell permeability so much that when similar permeabilization was simulated on uninfected red cells, lysis occurred before approximately 48 h. So how could infected cells, with a growing parasite inside, prevent lysis before the parasite has completed its developmental cycle? A mathematical model of the homeostasis of infected red cells suggested that it is the wasteful consumption of host cell hemoglobin that prevents early lysis by the progressive reduction in the colloid-osmotic pressure within the host (the colloid-osmotic hypothesis). However, two critical model predictions, that infected cells would swell to near prelytic sphericity and that the hemoglobin concentration would become progressively reduced, remained controversial. In this paper, we are able for the first time to correlate model predictions with recent experimental data in the literature and explore the fine details of the homeostasis of infected red blood cells during five model-defined periods of parasite development. The conclusions suggest that infected red cells do reach proximity to lytic rupture regardless of their actual volume, thus requiring a progressive reduction in their hemoglobin concentration to prevent premature lysis.

High-repetition-rate Combustion Thermometry with Two-line Atomic Fluorescence Excited by Diode Lasers

Optics Letters. Aug, 2009  |  Pubmed ID: 19684826

We report on kilohertz-repetition-rate flame temperature measurements performed using blue diode lasers. Two-line atomic fluorescence was performed by using diode lasers emitting at around 410 and 451 nm to probe seeded atomic indium. At a repetition rate of 3.5 kHz our technique offers a precision of 1.5% at 2000 K in laminar methane/air flames. The spatial resolution is better than 150 microm, while the setup is compact and easy to operate, at much lower cost than alternative techniques. By modeling the spectral overlap between the locked laser and the probed indium lines we avoid the need for any calibration of the measurements. We demonstrate the capability of the technique for time-resolved measurements in an acoustically perturbed flame. The technique is applicable in flames with a wide range of compositions including sooting flames.

Quantitative Fluorescence Microscopy Techniques

Methods in Molecular Biology (Clifton, N.J.). 2009  |  Pubmed ID: 19768427

Fluorescence microscopy is a non-invasive technique that allows high resolution imaging of cytoskeletal structures. Advances in the field of fluorescent labelling (e.g., fluorescent proteins, quantum dots, tetracystein domains) and optics (e.g., super-resolution techniques and quantitative methods) not only provide better images of the cytoskeleton, but also offer an opportunity to quantify the complex of molecular events that populate this highly organised, yet dynamic, structure.For instance, fluorescence lifetime imaging microscopy and Förster resonance energy transfer imaging allow mapping of protein-protein interactions; furthermore, techniques based on the measurement of photobleaching kinetics (e.g., fluorescence recovery after photobleaching, fluorescence loss in photobleaching, and fluorescence localisation after photobleaching) permit the characterisation of axonal transport and, more generally, diffusion of relevant biomolecules.Quantitative fluorescence microscopy techniques offer powerful tools for understanding the physiological and pathological roles of molecular machineries in the living cell.

Cavity Enhanced Spectroscopy of High-temperature H(2)o in the Near-infrared Using a Supercontinuum Light Source

Applied Spectroscopy. Dec, 2009  |  Pubmed ID: 20030985

In this paper we demonstrate how broadband cavity enhanced absorption spectroscopy (CEAS) with supercontinuum (SC) radiation in the near-infrared spectral range can be used as a sensitive, multiplexed, and simple tool to probe gas-phase species in high-temperature environments. Near-infrared SC radiation is generated by pumping a standard single-mode fiber with a picosecond fiber laser. Standard low reflectivity mirrors are used for the cavity and an optical spectrum analyzer is used for the detection of gas-phase species in combustion. The method is demonstrated by measuring flame generated H(2)O in the 1500 to 1550 nm region and room-temperature CO(2) between 1520 nm and 1660 nm. The broadband nature of the technique permits hundreds of rotational features to be recorded, giving good potential to unravel complex, convoluted spectra. We discuss practical issues concerning the implementation of the technique and present a straightforward method for calibration of the CEAS system via a cavity ringdown measurement. Despite the large spectral variation of SC radiation from pulse to pulse, it is shown that SC sources can offer good stability for CEAS where a large number of SC pulses are typically averaged.

Phi2FLIM: a Technique for Alias-free Frequency Domain Fluorescence Lifetime Imaging

Optics Express. Dec, 2009  |  Pubmed ID: 20052246

A new approach to alias-free wide-field fluorescence lifetime imaging in the frequency domain is demonstrated using a supercontinuum source for fluorescence excitation and a phase-modulated image intensifier for detection. This technique is referred to as phi-squared fluorescence lifetime imaging (phi(2)FLIM). The phase modulation and square-wave gating of the image intensifier eliminate aliasing by the effective suppression of higher harmonics. The ability to use picosecond excitation pulses without aliasing expands the range of excitation sources available for frequency-domain fluorescence lifetime imaging (fd-FLIM) and improves the modulation depth of conventional homodyne fd-FLIM measurements, which use sinusoidal intensity modulation of the excitation source. The phi(2)FLIM results are analyzed using AB-plots, which facilitate the identification of mono-exponential and multi-exponential fluorescence decays and provide measurements of the fluorophore fractions in two component mixtures. The rapid acquisition speed of the technique enables lifetime measurements in dynamic systems, such as temporally evolving samples and samples that are sensitive to photo-bleaching. Rapid phi(2)FLIM measurements are demonstrated by imaging the dynamic mixing of two different dye solutions at 5.5 Hz. The tunability of supercontinuum radiation enables excitation wavelength resolved FLIM measurements, which facilitates analysis of samples containing multiple fluorophores with different absorption spectra.

Imaging Pharmaceutical Tablets with Optical Coherence Tomography

Journal of Pharmaceutical Sciences. Jan, 2010  |  Pubmed ID: 19544370

Optical coherence tomography (OCT) is a recently developed optical technique that produces depth profiles of three-dimensional objects. It is a nondestructive interferometric method responding to refractive index variation in the sample under study and can reach a penetration depth of a few millimetres. OCT employs near-infrared (NIR) light and therefore provides a link between NIR spectroscopy and Terahertz (THz) measurements that are often used to characterise tablets. In this article we assess the potential of OCT as a reliable and practical tool in the analysis of pharmaceutical tablets and coatings. A variety of tablets were tested with different shapes, formulations and coatings. We consider the origins of contrast in the obtained images and demonstrate that it correlates strongly with the expected tablet structure. The influence of absorption and scattering are considered for the wavelength ranges used. The results show that OCT is a promising diagnostic tool with an important role to play in the tablet and coating technologies. The high measurement speed of OCT and its relative ease of implementation make it also an attractive candidate technology for in-line quality control during manufacturing.

Towards Multiparametric Fluorescent Imaging of Amyloid Formation: Studies of a YFP Model of Alpha-synuclein Aggregation

Journal of Molecular Biology. Jan, 2010  |  Pubmed ID: 19891973

Misfolding and aggregation of proteins are characteristics of a range of increasingly prevalent neurodegenerative disorders including Alzheimer's and Parkinson's diseases. In Parkinson's disease and several closely related syndromes, the protein alpha-synuclein (AS) aggregates and forms amyloid-like deposits in specific regions of the brain. Fluorescence microscopy using fluorescent proteins, for instance the yellow fluorescent protein (YFP), is the method of choice to image molecular events such as protein aggregation in living organisms. The presence of a bulky fluorescent protein tag, however, may potentially affect significantly the properties of the protein of interest; for AS in particular, its relative small size and, as an intrinsically unfolded protein, its lack of defined secondary structure could challenge the usefulness of fluorescent-protein-based derivatives. Here, we subject a YFP fusion of AS to exhaustive studies in vitro designed to determine its potential as a means of probing amyloid formation in vivo. By employing a combination of biophysical and biochemical studies, we demonstrate that the conjugation of YFP does not significantly perturb the structure of AS in solution and find that the AS-YFP protein forms amyloid deposits in vitro that are essentially identical with those observed for wild-type AS, except that they are fluorescent. Of the several fluorescent properties of the YFP chimera that were assayed, we find that fluorescence anisotropy is a particularly useful parameter to follow the aggregation of AS-YFP, because of energy migration Förster resonance energy transfer (emFRET or homoFRET) between closely positioned YFP moieties occurring as a result of the high density of the fluorophore within the amyloid species. Fluorescence anisotropy imaging microscopy further demonstrates the ability of homoFRET to distinguish between soluble, pre-fibrillar aggregates and amyloid fibrils of AS-YFP. Our results validate the use of fluorescent protein chimeras of AS as representative models for studying protein aggregation and offer new opportunities for the investigation of amyloid aggregation in vivo using YFP-tagged proteins.

Rotaviruses Associate with Cellular Lipid Droplet Components to Replicate in Viroplasms, and Compounds Disrupting or Blocking Lipid Droplets Inhibit Viroplasm Formation and Viral Replication

Journal of Virology. Jul, 2010  |  Pubmed ID: 20335253

Rotaviruses are a major cause of acute gastroenteritis in children worldwide. Early stages of rotavirus assembly in infected cells occur in viroplasms. Confocal microscopy demonstrated that viroplasms associate with lipids and proteins (perilipin A, ADRP) characteristic of lipid droplets (LDs). LD-associated proteins were also found to colocalize with viroplasms containing a rotaviral NSP5-enhanced green fluorescent protein (EGFP) fusion protein and with viroplasm-like structures in uninfected cells coexpressing viral NSP2 and NSP5. Close spatial proximity of NSP5-EGFP and cellular perilipin A was confirmed by fluorescence resonance energy transfer. Viroplasms appear to recruit LD components during the time course of rotavirus infection. NSP5-specific siRNA blocked association of perilipin A with NSP5 in viroplasms. Viral double-stranded RNA (dsRNA), NSP5, and perilipin A cosedimented in low-density gradient fractions of rotavirus-infected cell extracts. Chemical compounds interfering with LD formation (isoproterenol plus isobutylmethylxanthine; triacsin C) decreased the number of viroplasms and inhibited dsRNA replication and the production of infectious progeny virus; this effect correlated with significant protection of cells from virus-associated cytopathicity. Rotaviruses represent a genus of another virus family utilizing LD components for replication, pointing at novel therapeutic targets for these pathogens.

Detection of Plasmodium Falciparum-infected Red Blood Cells by Optical Stretching

Journal of Biomedical Optics. May-Jun, 2010  |  Pubmed ID: 20615000

We present the application of a microfluidic optical cell stretcher to measure the elasticity of malaria-infected red blood cells. The measurements confirm an increase in host cell rigidity during the maturation of the parasite Plasmodium falciparum. The device combines the selectivity and sensitivity of single-cell elasticity measurements with a throughput that is higher than conventional single-cell techniques. The method has potential to detect early stages of infection with excellent sensitivity and high speed.

Biophotonic Techniques for the Study of Malaria-infected Red Blood Cells

Medical & Biological Engineering & Computing. Oct, 2010  |  Pubmed ID: 20661776

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.

Sensitive Method for the Kinetic Measurement of Trace Species in Liquids Using Cavity Enhanced Absorption Spectroscopy with Broad Bandwidth Supercontinuum Radiation

Analytical Chemistry. Sep, 2010  |  Pubmed ID: 20672827

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.

Quantitative Imaging of Human Red Blood Cells Infected with Plasmodium Falciparum

Biophysical Journal. Aug, 2010  |  Pubmed ID: 20682274

During its 48 h asexual reproduction cycle, the malaria parasite Plasmodium falciparum ingests and digests hemoglobin in excess of its metabolic requirements and causes major changes in the homeostasis of the host red blood cell (RBC). A numerical model suggested that this puzzling excess consumption of hemoglobin is necessary for the parasite to reduce the colloidosmotic pressure within the host RBC, thus preventing lysis before completion of its reproduction cycle. However, the validity of the colloidosmotic hypothesis appeared to be compromised by initial conflicts between model volume predictions and experimental observations. Here, we investigated volume and membrane area changes in infected RBCs (IRBCs) using fluorescence confocal microscopy on calcein-loaded RBCs. Substantial effort was devoted to developing and testing a new threshold-independent algorithm for the precise estimation of cell volumes and surface areas to overcome the shortfalls of traditional methods. We confirm that the volume of IRBCs remains almost constant during parasite maturation, suggesting that the reported increase in IRBCs' osmotic fragility results from a reduction in surface area and increased lytic propensity on volume expansion. These results support the general validity of the colloidosmotic hypothesis, settle the IRBC volume debate, and help to constrain the range of parameter values in the numerical model.

Catalytic and Chaperone-like Functions in an Intrinsically Disordered Protein Associated with Desiccation Tolerance

Proceedings of the National Academy of Sciences of the United States of America. Sep, 2010  |  Pubmed ID: 20805515

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.

Hydrogen Ion Dynamics in Human Red Blood Cells

The Journal of Physiology. Dec, 2010  |  Pubmed ID: 20962000

Our understanding of pH regulation within red blood cells (RBCs) has been inferred mainly from indirect experiments rather than from in situ measurements of intracellular pH (pH(i)). The present work shows that carboxy-SNARF-1, a pH fluorophore, when used with confocal imaging or flow cytometry, reliably reports pH(i) in individual, human RBCs, provided intracellular fluorescence is calibrated using a 'null-point' procedure. Mean pH(i) was 7.25 in CO(2)/HCO(3)(-)-buffered medium and 7.15 in Hepes-buffered medium, and varied linearly with extracellular pH (slope of 0.77). Intrinsic (non-CO(2)/HCO(3)(-)-dependent) buffering power, estimated in the intact cell (85 mmol (l cell)(-1) (pH unit)(-1) at resting pH(i)), was somewhat higher than previous estimates from cell lysates (50-70 mmol (l cell)(-1) (pH unit)(-1)). Acute displacement of pH(i) (superfusion of weak acids/bases) triggered rapid pH(i) recovery. This was mediated via membrane Cl(-)/HCO(3)(-) exchange (the AE1 gene product), irrespective of whether recovery was from an intracellular acid or base load, and with no evident contribution from other transporters such as Na(+)/H(+) exchange. H(+)-equivalent flux through AE1 was a linear function of [H(+)](i) and reversed at resting pH(i), indicating that its activity is not allosterically regulated by pH(i), in contrast to other AE isoforms. By simultaneously monitoring pH(i) and markers of cell volume, a functional link between membrane ion transport, volume and pH(i) was demonstrated. RBC pH(i) is therefore tightly regulated via AE1 activity, but modulated during changes of cell volume. A comparable volume-pH(i) link may also be important in other cell types expressing anion exchangers. Direct measurement of pH(i) should be useful in future investigations of RBC physiology and pathology.

An Adaptive Filter for Studying the Life Cycle of Optical Rogue Waves

Optics Express. Dec, 2010  |  Pubmed ID: 21164960

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.

Predicting Supercontinuum Pulse Collisions with Simulations Exhibiting Temporal Aliasing

Optics Letters. Dec, 2010  |  Pubmed ID: 21165118

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.

A FRET Sensor for Non-invasive Imaging of Amyloid Formation in Vivo

Chemphyschem : a European Journal of Chemical Physics and Physical Chemistry. Feb, 2011  |  Pubmed ID: 21308945

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.

Theodor Förster: a Giant of Modern Photochemistry

Chemphyschem : a European Journal of Chemical Physics and Physical Chemistry. Feb, 2011  |  Pubmed ID: 21344585

HomoFRET Fluorescence Anisotropy Imaging As a Tool to Study Molecular Self-assembly in Live Cells

Chemphyschem : a European Journal of Chemical Physics and Physical Chemistry. Feb, 2011  |  Pubmed ID: 21344590

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.

Design and Application of a Confocal Microscope for Spectrally Resolved Anisotropy Imaging

Optics Express. Jan, 2011  |  Pubmed ID: 21369074

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.

X-ray Microanalysis Investigation of the Changes in Na, K, and Hemoglobin Concentration in Plasmodium Falciparum-infected Red Blood Cells

Biophysical Journal. Mar, 2011  |  Pubmed ID: 21402025

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.

Optical Detection of the Anesthetic Agent Propofol in the Gas Phase

Analytical Chemistry. May, 2011  |  Pubmed ID: 21526760

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.

In Situ Measurements of the Formation and Morphology of Intracellular β-amyloid Fibrils by Super-resolution Fluorescence Imaging

Journal of the American Chemical Society. Aug, 2011  |  Pubmed ID: 21793568

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.

Surface Enhanced Coherent Anti-stokes Raman Scattering on Nanostructured Gold Surfaces

Nano Letters. Dec, 2011  |  Pubmed ID: 22074256

Coherent anti-Stokes Raman spectroscopy (CARS) is a well-known tool in multiphoton imaging and nonlinear spectroscopy. In this work we combine CARS with plasmonic surface enhancement on reproducible nanostructured surfaces. We demonstrate strong correlation between plasmon resonances and surface-enhanced CARS (SECARS) intensities on our nanostructured surfaces and show that an enhancement of ∼10(5) can be obtained over standard CARS. Furthermore, we find SECARS to be >10(3) times more sensitive than surface-enhanced Raman Spectroscopy (SERS). We also demonstrate SECARS imaging of molecular monolayers. Our work paves the way for reliable single molecule Raman spectroscopy and fast molecular imaging on plasmonic surfaces.

Intrinsically Disordered Proteins As Molecular Shields

Molecular BioSystems. Jan, 2012  |  Pubmed ID: 21909508

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.

ALS Mutations in FUS Cause Neuronal Dysfunction and Death in Caenorhabditis Elegans by a Dominant Gain-of-function Mechanism

Human Molecular Genetics. Jan, 2012  |  Pubmed ID: 21949354

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.

Periodic Interactions Between Solitons and Dispersive Waves During the Generation of Non-coherent Supercontinuum Radiation

Optics Express. Mar, 2012  |  Pubmed ID: 22418513

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.

Analysis of the Native Structure, Stability and Aggregation of Biotinylated Human Lysozyme

PloS One. 2012  |  Pubmed ID: 23166837

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.

Protein Amyloids Develop an Intrinsic Fluorescence Signature During Aggregation

The Analyst. Apr, 2013  |  Pubmed ID: 23420088

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.

MBNL1 and PTB Cooperate to Repress Splicing of Tpm1 Exon 3

Nucleic Acids Research. May, 2013  |  Pubmed ID: 23511971

Exon 3 of the rat α-tropomyosin (Tpm1) gene is repressed in smooth muscle cells, allowing inclusion of the mutually exclusive partner exon 2. Two key types of elements affect repression of exon 3 splicing: binding sites for polypyrimidine tract-binding protein (PTB) and additional negative regulatory elements consisting of clusters of UGC or CUG motifs. Here, we show that the UGC clusters are bound by muscleblind-like proteins (MBNL), which act as repressors of Tpm1 exon 3. We show that the N-terminal region of MBNL1, containing its four CCCH zinc-finger domains, is sufficient to mediate repression. The same region of MBNL1 can make a direct protein-to-protein interaction with PTB, and RNA binding by MBNL promotes this interaction, apparently by inducing a conformational change in MBNL. Moreover, single molecule analysis showed that MBNL-binding sites increase the binding of PTB to its own sites. Our data suggest that the smooth muscle splicing of Tpm1 is mediated by allosteric assembly of an RNA-protein complex minimally comprising PTB, MBNL and their cognate RNA-binding sites.

Lifetime Imaging of a Fluorescent Protein Sensor Reveals Surprising Stability of ER Thiol Redox

The Journal of Cell Biology. Apr, 2013  |  Pubmed ID: 23589496

Interfering with disulfide bond formation impedes protein folding and promotes endoplasmic reticulum (ER) stress. Due to limitations in measurement techniques, the relationships of altered thiol redox and ER stress have been difficult to assess. We report that fluorescent lifetime measurements circumvented the crippling dimness of an ER-tuned fluorescent redox-responsive probe (roGFPiE), faithfully tracking the activity of the major ER-localized protein disulfide isomerase, PDI. In vivo lifetime imaging by time-correlated single-photon counting (TCSPC) recorded subtle changes in ER redox poise induced by exposure of mammalian cells to a reducing environment but revealed an unanticipated stability of redox to fluctuations in unfolded protein load. By contrast, TCSPC of roGFPiE uncovered a hitherto unsuspected reductive shift in the mammalian ER upon loss of luminal calcium, whether induced by pharmacological inhibition of calcium reuptake into the ER or by physiological activation of release channels. These findings recommend fluorescent lifetime imaging as a sensitive method to track ER redox homeostasis in mammalian cells.

A Label-free, Quantitative Assay of Amyloid Fibril Growth Based on Intrinsic Fluorescence

Chembiochem : a European Journal of Chemical Biology. May, 2013  |  Pubmed ID: 23592254

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.

Correcting Chromatic Offset in Multicolor Super-resolution Localization Microscopy

Optics Express. May, 2013  |  Pubmed ID: 23669954

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).

A Cancer-associated BRCA2 Mutation Reveals Masked Nuclear Export Signals Controlling Localization

Nature Structural & Molecular Biology. Oct, 2013  |  Pubmed ID: 24013206

Germline missense mutations affecting a single BRCA2 allele predispose humans to cancer. Here we identify a protein-targeting mechanism that is disrupted by the cancer-associated mutation, BRCA2(D2723H), and that controls the nuclear localization of BRCA2 and its cargo, the recombination enzyme RAD51. A nuclear export signal (NES) in BRCA2 is masked by its interaction with a partner protein, DSS1, such that point mutations impairing BRCA2-DSS1 binding render BRCA2 cytoplasmic. In turn, cytoplasmic mislocalization of mutant BRCA2 inhibits the nuclear retention of RAD51 by exposing a similar NES in RAD51 that is usually obscured by the BRCA2-RAD51 interaction. Thus, a series of NES-masking interactions localizes BRCA2 and RAD51 in the nucleus. Notably, BRCA2(D2723H) decreases RAD51 nuclear retention even when wild-type BRCA2 is also present. Our findings suggest a mechanism for the regulation of the nucleocytoplasmic distribution of BRCA2 and RAD51 and its impairment by a heterozygous disease-associated mutation.

High Sensitivity Liquid Phase Measurements Using Broadband Cavity Enhanced Absorption Spectroscopy (BBCEAS) Featuring a Low Cost Webcam Based Prism Spectrometer

The Analyst. Nov, 2013  |  Pubmed ID: 24049768

Cavity enhanced techniques enable high sensitivity absorption measurements in the liquid phase but are typically more complex, and much more expensive, to perform than conventional absorption methods. The latter attributes have so far prevented a wide spread use of these methods in the analytical sciences. In this study we demonstrate a novel BBCEAS instrument that is sensitive, yet simple and economical to set up and operate. We use a prism spectrometer with a low cost webcam as the detector in conjunction with an optical cavity consisting of two R = 0.99 dielectric mirrors and a white light LED source for illumination. High sensitivity liquid phase measurements were made on samples contained in 1 cm quartz cuvettes placed at normal incidence to the light beam in the optical cavity. The cavity enhancement factor (CEF) with water as the solvent was determined directly by phase shift cavity ring down spectroscopy (PS-CRDS) and also by calibration with Rhodamine 6G solutions. Both methods yielded closely matching CEF values of ~60. The minimum detectable change in absorption (αmin) was determined to be 6.5 × 10(-5) cm(-1) at 527 nm and was limited only by the 8 bit resolution of the particular webcam detector used, thus offering scope for further improvement. The instrument was used to make representative measurements on dye solutions and in the determination of nitrite concentrations in a variation of the widely used Griess Assay. Limits of detection (LOD) were ~850 pM for Rhodamine 6G and 3.7 nM for nitrite, respectively. The sensitivity of the instrument compares favourably with previous cavity based liquid phase studies whilst being achieved at a small fraction of the cost hitherto reported, thus opening the door to widespread use in the community. Further means of improving sensitivity are discussed in the paper.

A Quantitative Protocol for Intensity-based Live Cell FRET Imaging

Methods in Molecular Biology (Clifton, N.J.). 2014  |  Pubmed ID: 24108638

Förster resonance energy transfer (FRET) has become one of the most ubiquitous and powerful methods to quantify protein interactions in molecular biology. FRET refers to the sensitization of an acceptor molecule through transfer of energy from a nearby donor, and it can occur if the emission band of the donor exhibits spectral overlap with the absorption band of the acceptor molecule. Numerous methods exist to quantify FRET levels from interacting protein labels including fluorescence lifetime, acceptor photobleaching, and polarization-resolved imaging (Lakowicz, Principles of fluorescence spectroscopy, 2006; Jares-Erijman and Jovin, Curr Opin Chem Biol 10(5):409-416, 2006; van Munster and Gadella, Microscopy techniques, 2005). For live cell imaging, however, sensitized emission FRET (seFRET) is the most powerful and robust method of FRET signal quantification (Chakrabortee et al., Proc Natl Acad Sci USA 107(37):16084-16089, 2010). It is fast, can be applied using straight forward microscopy equipment, and offers information not only on strength of interaction but, uniquely, also on the relative changes between interacting and noninteracting moieties in the reaction, referred to as FRET stoichiometry. A rigorous and quantitative application of seFRET is far from trivial, however, and requires appropriate calibration experiments and constructs, control over hardware settings, and appropriate image processing steps.This protocol presents a rigorous method to perform quantitative seFRET measurements in live cells, providing the maximum possible information content from the measurement. The theoretical development and validation of the method is described in detail in Elder et al. (J R Soc Interface 6:S59-S81, 2009) where it is also demonstrated in the kinetic ("time-lapse") analysis of protein interactions governing mitosis. The present protocol gives a detailed recipe for application of seFRET. It is written specifically for use with CFP (cyan fluorescence protein) as donor fluorophore and YFP (yellow fluorescent protein) as acceptor fluorophore, a popular choice for many experiments. The protocol is however valid for any other FRET fluorophore pair, and we indicate how to adapt the protocol in such situations. We also provide a software program that automates the calibration tasks outlined in this protocol and which is available for free to download ( http://wiki.laser.ceb.cam.ac.uk/wiki/index.php/Resources ).

Extracellular Monomeric Tau Protein is Sufficient to Initiate the Spread of Tau Protein Pathology

The Journal of Biological Chemistry. Jan, 2014  |  Pubmed ID: 24235150

Understanding the formation and propagation of aggregates of the Alzheimer disease-associated Tau protein in vivo is vital for the development of therapeutics for this devastating disorder. Using our recently developed live-cell aggregation sensor in neuron-like cells, we demonstrate that different variants of exogenous monomeric Tau, namely full-length Tau (hTau40) and the Tau-derived construct K18 comprising the repeat domain, initially accumulate in endosomal compartments, where they form fibrillar seeds that subsequently induce the aggregation of endogenous Tau. Using superresolution imaging, we confirm that fibrils consisting of endogenous and exogenous Tau are released from cells and demonstrate their potential to spread Tau pathology. Our data indicate a greater pathological risk and potential toxicity than hitherto suspected for extracellular soluble Tau.

Direct Observation of Heterogeneous Amyloid Fibril Growth Kinetics Via Two-color Super-resolution Microscopy

Nano Letters. Jan, 2014  |  Pubmed ID: 24303845

The self-assembly of normally soluble proteins into fibrillar amyloid structures is associated with a range of neurodegenerative disorders, such as Parkinson's and Alzheimer's diseases. In the present study, we show that specific events in the kinetics of the complex, multistep aggregation process of one such protein, α-synuclein, whose aggregation is a characteristic hallmark of Parkinson's disease, can be followed at the molecular level using optical super-resolution microscopy. We have explored in particular the elongation of preformed α-synuclein fibrils; using two-color single-molecule localization microscopy we are able to provide conclusive evidence that the elongation proceeds from both ends of the fibril seeds. Furthermore, the technique reveals a large heterogeneity in the growth rates of individual fibrils; some fibrils exhibit no detectable growth, whereas others extend to more than ten times their original length within hours. These large variations in the growth kinetics can be attributed to fibril structural polymorphism. Our technique offers new capabilities in the study of amyloid growth dynamics at the molecular level and is readily translated to the study of the self-assembly of other nanostructures.

TestSTORM: Simulator for Optimizing Sample Labeling and Image Acquisition in Localization Based Super-resolution Microscopy

Biomedical Optics Express. Mar, 2014  |  Pubmed ID: 24688813

Localization-based super-resolution microscopy image quality depends on several factors such as dye choice and labeling strategy, microscope quality and user-defined parameters such as frame rate and number as well as the image processing algorithm. Experimental optimization of these parameters can be time-consuming and expensive so we present TestSTORM, a simulator that can be used to optimize these steps. TestSTORM users can select from among four different structures with specific patterns, dye and acquisition parameters. Example results are shown and the results of the vesicle pattern are compared with experimental data. Moreover, image stacks can be generated for further evaluation using localization algorithms, offering a tool for further software developments.

Direct Observations of Amyloid β Self-assembly in Live Cells Provide Insights into Differences in the Kinetics of Aβ(1-40) and Aβ(1-42) Aggregation

Chemistry & Biology. Jun, 2014  |  Pubmed ID: 24856820

Insight into how amyloid β (Aβ) aggregation occurs in vivo is vital for understanding the molecular pathways that underlie Alzheimer's disease and requires new techniques that provide detailed kinetic and mechanistic information. Using noninvasive fluorescence lifetime recordings, we imaged the formation of Aβ(1-40) and Aβ(1-42) aggregates in live cells. For both peptides, the cellular uptake via endocytosis is rapid and spontaneous. They are then retained in lysosomes, where their accumulation leads to aggregation. The kinetics of Aβ(1-42) aggregation are considerably faster than those of Aβ(1-40) and, unlike those of the latter peptide, show no detectable lag phase. We used superresolution fluorescence imaging to examine the resulting aggregates and could observe compact amyloid structures, likely because of spatial confinement within cellular compartments. Taken together, these findings provide clues as to how Aβ aggregation may occur within neurons.

Analyzing Receptor Assemblies in the Cell Membrane Using Fluorescence Anisotropy Imaging with TIRF Microscopy

PloS One. 2014  |  Pubmed ID: 24945870

Signaling within and between animal cells is controlled by the many receptor proteins in their membrane. They variously operate as trans-membrane monomers and homo- or hetero-dimers, and may assemble with ion-channels: analyses thereof are needed in studies of receptor actions in tissue physiology and pathology. Interactions between membrane proteins are detectable when pre-labeled with fluorophores, but a much fuller analysis is achievable via advanced optical techniques on living cells. In this context, the measurement of polarization anisotropy in the emitted fluorescence has been the least exploited. Here we demonstrate its methodology and particular advantages in the study of receptor protein assembly. Through excitation in both TIRF and EPI fluorescence illumination modes we are able to quantify and suppress contributions to the signal from extraneous intra-cellular fluorescence, and we show that the loss of fluorescence-polarization measured in membrane proteins reports on receptor protein assembly in real time. Receptor monomers and homo-dimers in the cell membrane can be analyzed quantitatively and for homo-dimers only a single fluorescent marker is needed, thus suppressing ambiguities that arise in alternative assays, which require multiple label moieties and which are thus subject to stoichiometric uncertainty.

Highly Potent Soluble Amyloid-β Seeds in Human Alzheimer Brain but Not Cerebrospinal Fluid

Brain : a Journal of Neurology. Nov, 2014  |  Pubmed ID: 25212850

The soluble fraction of brain samples from patients with Alzheimer's disease contains highly biologically active amyloid-β seeds. In this study, we sought to assess the potency of soluble amyloid-β seeds derived from the brain and cerebrospinal fluid. Soluble Alzheimer's disease brain extracts were serially diluted and then injected into the hippocampus of young, APP transgenic mice. Eight months later, seeded amyloid-β deposition was evident even when the hippocampus received subattomole amounts of brain-derived amyloid-β. In contrast, cerebrospinal fluid from patients with Alzheimer's disease, which contained more than 10-fold higher levels of amyloid-β peptide than the most concentrated soluble brain extracts, did not induce detectable seeding activity in vivo. Similarly, cerebrospinal fluid from aged APP-transgenic donor mice failed to induce cerebral amyloid-β deposition. In comparison to the soluble brain fraction, cerebrospinal fluid largely lacked N-terminally truncated amyloid-β species and exhibited smaller amyloid-β-positive particles, features that may contribute to the lack of in vivo seeding by cerebrospinal fluid. Interestingly, the same cerebrospinal fluid showed at least some seeding activity in an in vitro assay. The present results indicate that the biological seeding activity of soluble amyloid-β species is orders of magnitude greater in brain extracts than in the cerebrospinal fluid.

Retarded PDI Diffusion and a Reductive Shift in Poise of the Calcium Depleted Endoplasmic Reticulum

BMC Biology. 2015  |  Pubmed ID: 25575667

Endoplasmic reticulum (ER) lumenal protein thiol redox balance resists dramatic variation in unfolded protein load imposed by diverse physiological challenges including compromise in the key upstream oxidases. Lumenal calcium depletion, incurred during normal cell signaling, stands out as a notable exception to this resilience, promoting a rapid and reversible shift towards a more reducing poise. Calcium depletion induced ER redox alterations are relevant to physiological conditions associated with calcium signaling, such as the response of pancreatic cells to secretagogues and neuronal activity. The core components of the ER redox machinery are well characterized; however, the molecular basis for the calcium-depletion induced shift in redox balance is presently obscure.

Structural Analysis of Herpes Simplex Virus by Optical Super-resolution Imaging

Nature Communications. 2015  |  Pubmed ID: 25609143

Herpes simplex virus type-1 (HSV-1) is one of the most widespread pathogens among humans. Although the structure of HSV-1 has been extensively investigated, the precise organization of tegument and envelope proteins remains elusive. Here we use super-resolution imaging by direct stochastic optical reconstruction microscopy (dSTORM) in combination with a model-based analysis of single-molecule localization data, to determine the position of protein layers within virus particles. We resolve different protein layers within individual HSV-1 particles using multi-colour dSTORM imaging and discriminate envelope-anchored glycoproteins from tegument proteins, both in purified virions and in virions present in infected cells. Precise characterization of HSV-1 structure was achieved by particle averaging of purified viruses and model-based analysis of the radial distribution of the tegument proteins VP16, VP1/2 and pUL37, and envelope protein gD. From this data, we propose a model of the protein organization inside the tegument.

A Method to Quantify FRET Stoichiometry with Phasor Plot Analysis and Acceptor Lifetime Ingrowth

Biophysical Journal. Mar, 2015  |  Pubmed ID: 25762312

FRET is widely used for the study of protein-protein interactions in biological samples. However, it is difficult to quantify both the FRET efficiency (E) and the affinity (Kd) of the molecular interaction from intermolecular FRET signals in samples of unknown stoichiometry. Here, we present a method for the simultaneous quantification of the complete set of interaction parameters, including fractions of bound donors and acceptors, local protein concentrations, and dissociation constants, in each image pixel. The method makes use of fluorescence lifetime information from both donor and acceptor molecules and takes advantage of the linear properties of the phasor plot approach. We demonstrate the capability of our method in vitro in a microfluidic device and also in cells, via the determination of the binding affinity between tagged versions of glutathione and glutathione S-transferase, and via the determination of competitor concentration. The potential of the method is explored with simulations.

CYK4 Promotes Antiparallel Microtubule Bundling by Optimizing MKLP1 Neck Conformation

PLoS Biology. Apr, 2015  |  Pubmed ID: 25875822

Centralspindlin, a constitutive 2:2 heterotetramer of MKLP1 (a kinesin-6) and the non-motor subunit CYK4, plays important roles in cytokinesis. It is crucial for the formation of central spindle microtubule bundle structure. Its accumulation at the central antiparallel overlap zone is key for recruitment and regulation of downstream cytokinesis factors and for stable anchoring of the plasma membrane at the midbody. Both MKLP1 and CYK4 are required for efficient microtubule bundling. However, the mechanism by which CYK4 contributes to this is unclear. Here we performed structural and functional analyses of centralspindlin using high-speed atomic force microscopy, Fӧrster resonance energy transfer analysis, and in vitro reconstitution. Our data reveal that CYK4 binds to a globular mass in the atypically long MKLP1 neck domain between the catalytic core and the coiled coil and thereby reconfigures the two motor domains in the MKLP1 dimer to be suitable for antiparallel microtubule bundling. Our work provides insights into the microtubule bundling during cytokinesis and into the working mechanisms of the kinesins with non-canonical neck structures.

Fast Imaging of Live Organisms with Sculpted Light Sheets

Scientific Reports. 2015  |  Pubmed ID: 25893952

Light-sheet microscopy is an increasingly popular technique in the life sciences due to its fast 3D imaging capability of fluorescent samples with low photo toxicity compared to confocal methods. In this work we present a new, fast, flexible and simple to implement method to optimize the illumination light-sheet to the requirement at hand. A telescope composed of two electrically tuneable lenses enables us to define thickness and position of the light-sheet independently but accurately within milliseconds, and therefore optimize image quality of the features of interest interactively. We demonstrated the practical benefit of this technique by 1) assembling large field of views from tiled single exposure each with individually optimized illumination settings; 2) sculpting the light-sheet to trace complex sample shapes within single exposures. This technique proved compatible with confocal line scanning detection, further improving image contrast and resolution. Finally, we determined the effect of light-sheet optimization in the context of scattering tissue, devising procedures for balancing image quality, field of view and acquisition speed.

Visualizing Electromagnetic Fields at the Nanoscale by Single Molecule Localization

Nano Letters. May, 2015  |  Pubmed ID: 25915093

Coupling of light to the free electrons at metallic surfaces allows the confinement of electric fields to subwavelength dimensions, far below the optical diffraction limit. While this is routinely used to manipulate light at the nanoscale, in electro-optic devices and enhanced spectroscopic techniques, no characterization technique for imaging the underlying nanoscopic electromagnetic fields exists, which does not perturb the field or employ complex electron beam imaging. Here, we demonstrate the direct visualization of electromagnetic fields on patterned metallic substrates at nanometer resolution, exploiting a strong "autonomous" fluorescence-blinking behavior of single molecules within the confined fields allowing their localization. Use of DNA-constructs for precise positioning of fluorescence dyes on the surface induces this distance-dependent autonomous blinking thus completely obviating the need for exogenous agents or switching methods. Mapping such electromagnetic field distributions at nanometer resolution aids the rational design of nanometals for diverse photonic applications.

Fast and Simple Spectral FLIM for Biochemical and Medical Imaging

Optics Express. Sep, 2015  |  Pubmed ID: 26368450

Spectrally resolved fluorescence lifetime imaging microscopy (λFLIM) has powerful potential for biochemical and medical imaging applications. However, long acquisition times, low spectral resolution and complexity of λFLIM often narrow its use to specialized laboratories. Therefore, we demonstrate here a simple spectral FLIM based on a solid-state detector array providing in-pixel histrogramming and delivering faster acquisition, larger dynamic range, and higher spectral elements than state-of-the-art λFLIM. We successfully apply this novel microscopy system to biochemical and medical imaging demonstrating that solid-state detectors are a key strategic technology to enable complex assays in biomedical laboratories and the clinic.

In Situ Visualization of Block Copolymer Self-Assembly in Organic Media by Super-Resolution Fluorescence Microscopy

Chemistry (Weinheim an Der Bergstrasse, Germany). Dec, 2015  |  Pubmed ID: 26477697

Analytical methods that enable visualization of nanomaterials derived from solution self-assembly processes in organic solvents are highly desirable. Herein, we demonstrate the use of stimulated emission depletion microscopy (STED) and single molecule localization microscopy (SMLM) to map living crystallization-driven block copolymer (BCP) self-assembly in organic media at the sub-diffraction scale. Four different dyes were successfully used for single-colour super-resolution imaging of the BCP nanostructures allowing micelle length distributions to be determined in situ. Dual-colour SMLM imaging was used to measure and compare the rate of addition of red fluorescent BCP to the termini of green fluorescent seed micelles to generate block comicelles. Although well-established for aqueous systems, the results highlight the potential of super-resolution microscopy techniques for the interrogation of self-assembly processes in organic media.

ALS/FTD Mutation-Induced Phase Transition of FUS Liquid Droplets and Reversible Hydrogels into Irreversible Hydrogels Impairs RNP Granule Function

Neuron. Nov, 2015  |  Pubmed ID: 26526393

The mechanisms by which mutations in FUS and other RNA binding proteins cause ALS and FTD remain controversial. We propose a model in which low-complexity (LC) domains of FUS drive its physiologically reversible assembly into membrane-free, liquid droplet and hydrogel-like structures. ALS/FTD mutations in LC or non-LC domains induce further phase transition into poorly soluble fibrillar hydrogels distinct from conventional amyloids. These assemblies are necessary and sufficient for neurotoxicity in a C. elegans model of FUS-dependent neurodegeneration. They trap other ribonucleoprotein (RNP) granule components and disrupt RNP granule function. One consequence is impairment of new protein synthesis by cytoplasmic RNP granules in axon terminals, where RNP granules regulate local RNA metabolism and translation. Nuclear FUS granules may be similarly affected. Inhibiting formation of these fibrillar hydrogel assemblies mitigates neurotoxicity and suggests a potential therapeutic strategy that may also be applicable to ALS/FTD associated with mutations in other RNA binding proteins.

Optical Super-Resolution Imaging of β-Amyloid Aggregation In Vitro and In Vivo: Method and Techniques

Methods in Molecular Biology (Clifton, N.J.). 2016  |  Pubmed ID: 26235063

Super-resolution microscopy has emerged as a powerful and non-invasive tool for the study of molecular processes both in vitro and in live cells. In particular, super-resolution microscopy has proven valuable for research studies in protein aggregation. In this chapter we present details of recent advances in this method and the specific techniques, enabling the study of amyloid beta aggregation optically, both in vitro and in cells. First, we show that variants of optical super-resolution microscopy provide a capability to visualize oligomeric and fibrillar structures directly, providing detailed information on species morphology in vitro and even in situ, in the cellular environment. We focus on direct Stochastic Optical Reconstruction Microscopy, dSTORM, which provides morphological detail on spatial scales below 20 nm, and provide detailed protocols for its implementation in the context of amyloid beta research. Secondly, we present a range of optical techniques that offer super-resolution indirectly, which we call multi-parametric microscopy. The latter offers molecular scale information on self-assembly reactions via changes in protein or fluorophore spectral signatures. These techniques are empowered by our recent discovery that disease related amyloid proteins adopt intrinsic energy states upon fibrilisation. We show that fluorescence lifetime imaging provides a particularly sensitive readout to report on the aggregation state, which is robustly quantifiable for experiments performed either in vitro or in vivo.

HSV-1 Glycoproteins Are Delivered to Virus Assembly Sites Through Dynamin-Dependent Endocytosis

Traffic (Copenhagen, Denmark). Jan, 2016  |  Pubmed ID: 26459807

Herpes simplex virus-1 (HSV-1) is a large enveloped DNA virus that belongs to the family of Herpesviridae. It has been recently shown that the cytoplasmic membranes that wrap the newly assembled capsids are endocytic compartments derived from the plasma membrane. Here, we show that dynamin-dependent endocytosis plays a major role in this process. Dominant-negative dynamin and clathrin adaptor AP180 significantly decrease virus production. Moreover, inhibitors targeting dynamin and clathrin lead to a decreased transport of glycoproteins to cytoplasmic capsids, confirming that glycoproteins are delivered to assembly sites via endocytosis. We also show that certain combinations of glycoproteins colocalize with each other and with the components of clathrin-dependent and -independent endocytosis pathways. Importantly, we demonstrate that the uptake of neutralizing antibodies that bind to glycoproteins when they become exposed on the cell surface during virus particle assembly leads to the production of non-infectious HSV-1. Our results demonstrate that transport of viral glycoproteins to the plasma membrane prior to endocytosis is the major route by which these proteins are localized to the cytoplasmic virus assembly compartments. This highlights the importance of endocytosis as a major protein-sorting event during HSV-1 envelopment.

Proton Transfer and Structure-Specific Fluorescence in Hydrogen Bond-Rich Protein Structures

Journal of the American Chemical Society. Mar, 2016  |  Pubmed ID: 26824778

Protein structures which form fibrils have recently been shown to absorb light at energies in the near UV range and to exhibit a structure-specific fluorescence in the visible range even in the absence of aromatic amino acids. However, the molecular origin of this phenomenon has so far remained elusive. Here, we combine ab initio molecular dynamics simulations and fluorescence spectroscopy to demonstrate that these intrinsically fluorescent protein fibrils are permissive to proton transfer across hydrogen bonds which can lower electron excitation energies and thereby decrease the likelihood of energy dissipation associated with conventional hydrogen bonds. The importance of proton transfer on the intrinsic fluorescence observed in protein fibrils is signified by large reductions in the fluorescence intensity upon either fully protonating, or deprotonating, the fibrils at pH = 0 or 14, respectively. Thus, our results point to the existence of a structure-specific fluorophore that does not require the presence of aromatic residues or multiple bond conjugation that characterize conventional fluorescent systems. The phenomenon may have a wide range of implications in biological systems and in the design of self-assembled functional materials.

Nanoscopic Insights into Seeding Mechanisms and Toxicity of α-synuclein Species in Neurons

Proceedings of the National Academy of Sciences of the United States of America. Mar, 2016  |  Pubmed ID: 26993805

New strategies for visualizing self-assembly processes at the nanoscale give deep insights into the molecular origins of disease. An example is the self-assembly of misfolded proteins into amyloid fibrils, which is related to a range of neurodegenerative disorders, such as Parkinson's and Alzheimer's diseases. Here, we probe the links between the mechanism of α-synuclein (AS) aggregation and its associated toxicity by using optical nanoscopy directly in a neuronal cell culture model of Parkinson's disease. Using superresolution microscopy, we show that protein fibrils are taken up by neuronal cells and act as prion-like seeds for elongation reactions that both consume endogenous AS and suppress its de novo aggregation. When AS is internalized in its monomeric form, however, it nucleates and triggers the aggregation of endogenous AS, leading to apoptosis, although there are no detectable cross-reactions between externally added and endogenous protein species. Monomer-induced apoptosis can be reduced by pretreatment with seed fibrils, suggesting that partial consumption of the externally added or excess soluble AS can be significantly neuroprotective.

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