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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., (© 2013 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA. This is an open access article under the terms of the Attribution Non-Commercial NoDerivs License, which permits use and distribution in any medium, provided the original work is properly cited, the use is non-commercial and no modifications or adaptations are made.)

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.

Fluorescence lifetime imaging (FLIM) allows the quantification of sub‐cellular processes in situ, in living cells. A number of approaches have been developed to extract the lifetime from time‐domain FLIM data, but they are often limited in terms of speed, photon efficiency, precision or the dynamic range of lifetimes they can measure. Here, we focus on one of the best performing methods in the field, the centre‐of‐mass method (CMM), that conveys advantages in terms of speed and photon efficiency over others. In this paper, however, we identify a loss of photon efficiency of CMM for short lifetimes when background noise is present. We subsequently present a new development and generalization of CMM that provides for the rapid and accurate extraction of fluorescence lifetime over a large lifetime dynamic range. We provide software tools to simulate, validate and analyse FLIM data sets and compare the performance of our approach against the standard CMM and the commonly employed least‐square minimization (LSM) methods. Our method features a better photon efficiency than standard CMM and LSM and is robust in the presence of background noise. The algorithm is applicable to any time‐domain FLIM data set. [ABSTRACT FROM AUTHOR]

Immunofluorescence microscopy is routinely used in the diagnosis of and research on renal impairments. However, this highly specific technique is restricted in its maximum resolution to about 250 nm in the lateral and 700 nm in the axial directions and thus not sufficient to investigate the fine subcellular structure of the kidney's glomerular filtration barrier. In contrast, electron microscopy offers high resolution, but this comes at the cost of poor preservation of immunogenic epitopes and antibody penetration alongside a low throughput. Many of these drawbacks were overcome with the advent of super-resolution microscopy methods. So far, four different super-resolution approaches have been used to study the kidney: single-molecule localization microscopy (SMLM), stimulated emission depletion (STED) microscopy, structured illumination microscopy (SIM), and expansion microscopy (ExM), however, using different preservation methods and widely varying labelling strategies. In this work, all four methods were applied and critically compared on kidney slices obtained from samples treated with the most commonly used preservation technique: fixation by formalin and embedding in paraffin (FFPE). Strengths and weaknesses, as well as the practicalities of each method, are discussed to enable users of super-resolution microscopy in renal research make an informed decision on the best choice of technique. The methods discussed enable the efficient investigation of biopsies stored in kidney banks around the world. [ABSTRACT FROM AUTHOR]

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 developments to the technique reported here were essential to achieve the accuracy (±41K) and precision (±8K) 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 thermometry techniques perform poorly. We thus demonstrate a practical application of diode laser TLAF to a burner 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 O2, N2 and CH4 at fuel equivalence ratio of 2.32 and pressures ranging from 18.7 to 26.7kPa. 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 operating pressures. The data will contribute to modelling efforts to understand the surprisingly strong dependence 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 profile 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. [ABSTRACT FROM AUTHOR]

Abstract: 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. [Copyright &y& Elsevier]

Abstract: The effects of curvature and wrinkling on the growth of turbulent premixed flame kernels have been investigated using both 2D OH planar laser-induced fluorescence (PLIF) and 3D direct numerical simulation (DNS). Comparisons of results between the two approaches show a high level of agreement, providing confidence in the simplified chemistry treatment employed in the DNS, and indicating that chemistry may have only a limited influence on the evolution of the freely propagating flame. This is in contrast to previous studies of the very early flame development where chemistry may be dominant. Statistics for curvature and wrinkling are presented in the form of probability density functions, and there is good agreement with previous findings. The limitations of 2D PLIF measurements of curvature are quantified by comparison with full 3D information obtained from the DNS. The usefulness of PLIF in providing data over a wide parameter range is illustrated using statistics obtained from both CH4/air and H2/air mixtures, which show a markedly different behaviour due to their different thermo-diffusive properties. The results provide a demonstration of the combined power of PLIF and DNS for flame investigation. Each technique is shown to compensate for the weaknesses of the other and to reinforce the strengths of both. [Copyright &y& Elsevier]

Summary: 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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Abstract: 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 α-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. [Copyright &y& Elsevier]

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. [ABSTRACT FROM AUTHOR]

Abstract: The pH mediated intra-molecular association and inter-molecular aggregation of a range of amphiphilic poly(l-lysine iso-phthalamide) polymers have been investigated in aqueous solution over a range of pH values and concentrations. The desired functionality of these novel bioresponsive amphiphilic polymers was achieved by incorporating pendant hydrophilic carboxyl groups along the polymer backbone, via the l-lysine moiety, balanced by a degree of hydrophobicity introduced via the iso-phthaloyl moiety. Incorporation of low levels of bis-functional Cy3 (poly-Cy3) and/or Cy5 dye (poly-Cy3/5 or poly-Cy5) co-monomers in the responsive polymer backbone allowed detailed probing of the pH mediated hydrophobic association using a combination of optical spectroscopic techniques. Both steady-state fluorescence spectroscopy and fluorescence lifetime measurements of poly-Cy3 revealed a conformational transition at pH 4.5. Thus, below a critical pH the polymer collapsed into a compact globular structure (hypercoil) bringing the fluorophore molecules into close proximity with one another. This resulted in a dramatic reduction in fluorescence intensity and fluorescent lifetime in the single fluorophore systems (poly-Cy3) accompanied by a red shift in the maximum emission wavelength. Observed redshifts in the emission maxima and enhancements of fluorescent lifetimes with increasing polymer concentration suggested the formation of polymer aggregates. Fluorescence resonance energy transfer (FRET) was measured in mixtures of single fluorophore containing poly-Cy3 (donor) and poly-Cy5 (acceptor) and dual fluorophore containing poly-Cy3 (donor)/Cy5 (acceptor) in an effort to distinguish between intra-molecular versus inter-molecular association. The relevance of the results with respect to potential in vivo applications (drug delivery and biodiagnostics) is discussed. [Copyright &y& Elsevier]