A tomographic technique for the simultaneous imaging of temperature, chemical species, and pressure in reactive flows using absorption spectroscopy with frequency-agile lasers

This paper proposes a technique that can simultaneously retrieve distributions of temperature, concentration of chemical species, and pressure based on broad bandwidth, frequency-agile tomographic absorption spectroscopy. The technique holds particular promise for the study of dynamic combusting flows. A proof-of-concept numerical demonstration is presented, using representative phantoms to model conditions typically prevailing in near-atmospheric or high pressure flames. The simulations reveal both the feasibility of the proposed technique and its robustness. Our calculations indicate precisions of ∼70 K at flame temperatures and ∼0.05 bars at high pressure from reconstructions featuring as much as 5% Gaussian noise in the projections.This work was supported by the Seventh Framework Program (Grant Agreement No. PIIF-GA-2012-330840) of the European Union and was performed using the Darwin Supercomputer of the University of Cambridge High Performance Computing Service.Copyright 2014 American Institute of Physics. This article may be downloaded for personal use only. Any other use requires prior permission of the author and the American Institute of Physics. The article appeared in Applied Physics Letters 104, 034101 (2014) and may be found at (http://scitation.aip.org/content/aip/journal/apl/104/3/10.1063/1.4862754)

A numerical investigation of high-resolution multispectral absorption tomography for flow thermometry

Multispectral absorption tomography (MAT) is now a well-established technique that can be applied for the simultaneous imaging of temperature, species concentration, and pressure of reactive flows. However, only intermediate spatial resolution, on order of 15×15 grid points, has so far been achievable in previous demonstrations. The aim of the present work is to provide a numerical validation of our MAT algorithm for thermometry of combusting flows, but with greatly improved spatial resolution to motivate its experimental realization in practical environments. We demonstrate a grid resolution that is comparable to that of classical absorption tomography (CAT) containing 80×80 elements from only two orthogonal projections, which is impractical to realize with CAT but especially desirable for applications where optical access is limited. This is achieved using the smoothness assumption, which holds true under most combustion conditions. The study shows that better spatial resolution can be obtained through a simple increase in the spatial sampling frequency for the two available projections, as the smoothness condition becomes more reliable on smaller spatial scales. Our work also demonstrates the first application of MAT for full volumetric reconstructions. The studies thus provide robust guidelines for the implementation of MAT over large spatial scales and lay solid foundations for its development and application in complex technical combustion scenarios, where spatial resolution is crucial to investigate the interaction of flow phenomena with chemical reactions.This work was funded by the European Commission under Grant No. ASHTCSC 330840 and was partly performed using the Darwin Supercomputer of the University of Cambridge High Performance Computing Service. Clemens F. Kaminski also wishes to acknowledge EPSRC for funding (grant EP/L015889/1).This is the final published version of a paper published in Applied Physics B, February 2015, DOI 10.1007/s00340-015-6012-

Super-resolution imaging of alpha-synuclein polymorphisms and their potential role in neurodegeneration

The conversion of soluble, functional proteins into amyloid fibrils has been linked to the development of neurodegenerative disorders, including Parkinson's and Alzheimer's disease. In the brains of patients with these disorders, the increasing presence of amyloid-containing plaques corresponds to neuronal cell death and the worsening of symptoms. However, protein amyloids are not merely confined to dying cells. Rather, some show a propensity to be transmitted to, and enter adjacent cells and induce the polymerization of the native monomer population. Whether this process is directly associated with toxicity or not is still highly debated. In this mini review, we will discuss structural polymorphisms of α-synuclein, as determined by super-resolution imaging techniques, and how these may be related to neuronal toxicity.This work was funded by grants from the UK Medical Research Council (MR/K015850/1 and MR/K02292X/1), Alzheimer’s Research UK (ARUK-EG2012A-1), the UK Engineering and Physical Sciences Research Council (EP/H018301/1), and the Wellcome Trust (089703/ Z/09/Z)

Frontiers in structured illumination microscopy

© 2016 Optical Society of America. At the start of this millennium, the principles of structured illumination microscopy (SIM) had been established and the concept of resolution doubling demonstrated experimentally in two dimensions. Breathtaking advances have since taken place, making SIM one of the most powerful and versatile super resolution methods available today, routinely used in the study of biochemical processes in laboratories around the world. In theory there is no inherent limit to the resolution obtainable with certain modalities of SIM, and new variants have the potential to operate at even higher speeds and sensitivity than currently realized. In this review, we focus on the very latest innovations in SIM theory and practice, which are set to continue the revolution of this method into the future. Examples include confocal implementations of the SIM principle, which can be used in combination with two-photon excitation and adaptive optics. We present recent applications of such approaches in the life sciences, which illustrate their potential to revolutionize intravital research, by providing the ability to watch life at the molecular scale, at high speeds, and deep within living organisms. A different variant makes use of standing plasmonic waves or localized surface plasmons to confer performance enhancements to 2D SIM modalities. Research on these latter techniques is in its infancy but already shows great potential for their development into powerful in vitro probes for chemical processes at solid/liquid interfaces. Physical concepts are reviewed in detail, and future directions are presented along which the field might fruitfully develop, holding promise for new discoveries on the molecular scale.This work was supported by grants from the Leverhulme Trust, the Engineering and Physical Sciences Research Council, UK (grant EP/H018301/1), by the Medical Research Council (grant MR/K015850/1, and MR/K02292X/1), the Wellcome Trust (089703/Z/09/Z) and the Alzheimer Research UK Trust (ARUK-EG2012A-1)

Speed limits of structured illumination microscopy

A theoretical framework for widefield structured illumination microscopy (SIM) reconstruction from fewer than the commonly used nine raw frame acquisitions is introduced and applied in silico and in vitro. The proposed scheme avoids the recording of redundant spatial frequency components, which was necessary in previous SIM algorithms. This allows for gentler superresolution imaging at faster speeds. A doubling of frame rates is possible solely via changes in the computational reconstruction procedure. Furthermore, we explore numerically the effect of the sample movement on the reconstruction quality and the number of raw frames recordable. Our results show that there exists a limit above which deconvolution microscopy becomes superior to SIM.Engineering and Physical Sciences Research Council (EPSRC) (EP/H018301/1); Medical Research Council (MRC) (MR/K015850/1, MR/K02292X/1); Wellcome Trust (089703/Z/09/Z)

Blind assessment of localisation microscope image resolution

Abstract Background This paper analyses the resolution achieved in localisation microscopy experiments. The resolution is an essential metric for the correct interpretation of super-resolution images, but it varies between specimens due to different localisation precisions and densities. Methods By analysing localisation microscopy as a statistical method of Density Estimation, we present a method that produces a blind estimate of the resolution in a super-resolved image. This estimate is derived directly from the raw image data without the need for comparisons with known calibration specimens. It is corroborated with simulated and experimental data. Results and discussion Localisation microscopy has a resolution limit equal to 2σ, where σ is the r.m.s. localisation precision, evaluated as an average Thompson precision, Cramer Rao bound, or otherwise. Further, for a limited-sampling case in which there is only one localisation per fluorophore, the expected resolution of an optimised super-resolution image is worsened to approximately 3σ, due to smoothing processes that are necessarily involved in visualising the specimen with limited data. This 2σ or 3σ resolution can be estimated for any localisation microscopy specimen, and this metric can corroborate or replace empirical estimates of resolution. Other quantifiable resolution losses arise from sparse labelling, fluorescent label size, and motion blur.RIGHTS : This article is licensed under the BioMed Central licence at http://www.biomedcentral.com/about/license which is similar to the 'Creative Commons Attribution Licence'. In brief you may : copy, distribute, and display the work; make derivative works; or make commercial use of the work - under the following conditions: the original author must be given credit; for any reuse or distribution, it must be made clear to others what the license terms of this work are

Investigating State Restriction in Fluorescent Protein FRET Using Time-Resolved Fluorescence and Anisotropy

Most fluorescent proteins exhibit multiexponential fluorescence decays, indicating a heterogeneous excited state population. FRET between fluorescent proteins should therefore involve multiple energy transfer pathways. We recently demonstrated the FRET pathways between EGFP and mCherry (mC), upon the dimerization of 3-phosphoinositide dependent protein kinase 1 (PDK1), to be highly restricted. A mechanism for FRET restriction based on a highly unfavorable κ(2) orientation factor arising from differences in donor-acceptor transition dipole moment angles in a far from coplanar and near static interaction geometry was proposed. Here this is tested via FRET to mC arising from the association of glutathione (GSH) and glutathione S-transferase (GST) with an intrinsically homogeneous and more mobile donor Oregon Green 488 (OG). A new analysis of the acceptor window intensity, based on the turnover point of the sensitized fluorescence, is combined with donor window intensity and anisotropy measurements which show that unrestricted FRET to mC takes place. However, a long-lived anisotropy decay component in the donor window reveals a GST-GSH population in which FRET does not occur, explaining previous discrepancies between quantitative FRET measurements of GST-GSH association and their accepted values. This reinforces the importance of the local donor-acceptor environment in mediating energy transfer and the need to perform spectrally resolved intensity and anisotropy decay measurements in the accurate quantification of fluorescent protein FRET

Total internal reflection fluorescence anisotropy imaging microscopy: setup, calibration, and data processing for protein polymerization measurements in living cells

Fluorescence anisotropy imaging microscopy (FAIM) measures the depolarization properties of fluorophores to deduce molecular changes in their environment. For successful FAIM, several design principles have to be considered and a thorough system-specific calibration protocol is paramount. One important calibration parameter is the G factor, which describes the system-induced errors for different polarization states of light. The determination and calibration of the G factor is discussed in detail in this article. We present a novel measurement strategy, which is particularly suitable for FAIM with high numerical aperture objectives operating in TIRF illumination mode. The method makes use of evanescent fields that excite the sample with a polarization direction perpendicular to the image plane. Furthermore, we have developed an ImageJ/Fiji plugin, AniCalc, for FAIM data processing. We demonstrate the capabilities of our TIRF-FAIM system by measuring β-actin polymerization in human embryonic kidney cells and in retinal neurons.This work was funded by grants from the Medical Research Council UK (MR/K015850/1 and MR/K02292X/1), the EPSRC (EP/L015889/1 and EP/H018301/1), theWellcome Trust (3-3249/Z/16/Z and 089703/Z/09/Z), and In nitus, China, Ltd (CFK); the Cambridge Trust, Croucher Foundation, Sir Edward Youde Memorial Fund (HHWW); a Wellcome Trust Programme Grant (085314/Z/08/Z) and an ERC Advanced Grant (322817) (CEH)

Correlative STED and Atomic Force Microscopy on Live Astrocytes Reveals Plasticity of Cytoskeletal Structure and Membrane Physical Properties during Polarized Migration

The plasticity of the cytoskeleton architecture and membrane properties is important for the establishment of cell polarity, adhesion and migration. Here, we present a method which combines stimulated emission depletion (STED) super-resolution imaging and atomic force microscopy (AFM) to correlate cytoskeletal structural information with membrane physical properties in live astrocytes. Using STED compatible dyes for live cell imaging of the cytoskeleton, and simultaneously mapping the cell surface topology with AFM, we obtain unprecedented detail of highly organized networks of actin and microtubules in astrocytes. Combining mechanical data from AFM with optical imaging of actin and tubulin further reveals links between cytoskeleton organization and membrane properties. Using this methodology we illustrate that scratch-induced migration induces cytoskeleton remodeling. The latter is caused by a polarization of actin and microtubule elements within astroglial cell processes, which correlates strongly with changes in cell stiffness. The method opens new avenues for the dynamic probing of the membrane structural and functional plasticity of living brain cells. It is a powerful tool for providing new insights into mechanisms of cell structural remodeling during physiological or pathological processes, such as brain development or tumorigenesis.This work was supported by grants from College de France and ERC to NR, Paris 6 University doctoral school ED3C and Labex Memolife to GG. CFK acknowledges funding from the Engineering and Physical Sciences Research council (EPSRC, UK), the Wellcome Trust, UK, the Medical Research Council (MRC, UK) and Infinitus Ltd. GSKS acknowledges funding from the Wellcome Trust, UK and the MRC

Heparin acts as a structural component of β-endorphin amyloid fibrils rather than a simple aggregation promoter.

The aggregation promoter heparin is commonly used to study the aggregation kinetics and biophysical properties of protein amyloids. However, the underlying mechanism for amyloid promotion by heparin remains poorly understood. In the case of the neuropeptide β-endorphin that can reversibly adopt a functional amyloid form in nature, aggregation in the presence of heparin leads to a loss of function. Applying correlative optical super-resolution microscopy methods, we show that heparin incorporates into emerging β-endorphin fibrils forming an integral component and is essential for amyloid templating. This will have direct implications on β-endorphin's normal physiological function and raises concerns on the biological relevance of heparin-promoted amyloid models.This work was funded by grants from the Wellcome Trust, the Medical Research Council UK, the Alzheimer Research UK Trust, the Engineering and Physical Sciences Research Council UK, and the Biotechnology and Biological Sciences Research Council. NN was supported through Early PostDoc.Mobility personal fellowship from Swiss National Science Foundation