Time Resolution Of Seconds Research Articles

A high-precision view of intercompartmental drug transport via simultaneous, seconds-resolved, in situ measurements in the vein and brain.

The ability to measure specific molecules at multiple sites within the body simultaneously, and with a time resolution of seconds, could greatly advance our understanding of drug transport and elimination. As a proof-of-principle demonstration, here we describe the use of electrochemical aptamer-based (EAB) sensors to measure transport of the antibiotic vancomycin from the plasma (measured in the jugular vein) to the cerebrospinal fluid (measured in the lateral ventricle) of live rats with temporal resolution of a few seconds. In our first efforts, we made measurements solely in the ventricle. Doing so we find that, although the collection of hundreds of concentration values over a single drug lifetime enables high-precision estimates of the parameters describing intracranial transport, due to a mathematical equivalence, the data produce two divergent descriptions of the drug's plasma pharmacokinetics that fit the in-brain observations equally well. The simultaneous collection of intravenous measurements, however, resolves this ambiguity, enabling high-precision (typically of ±5 to ±20% at 95% confidence levels) estimates of the key pharmacokinetic parameters describing transport from the blood to the cerebrospinal fluid in individual animals. The availability of simultaneous, high-density 'in-vein' (plasma) and 'in-brain' (cerebrospinal fluid) measurements provides unique opportunities to explore the assumptions almost universally employed in earlier compartmental models of drug transport, allowing the quantitative assessment of, for example, the pharmacokinetic effects of physiological processes such as the bulk transport of the drug out of the CNS via the dural venous sinuses.

Spatiotemporal operando UV–vis spectroscopy: Development and mechanistic alternation of CO oxidation on Pt/Al2O3 on the reactor scale

Operando methodologies are widely used in heterogenous catalysis to understand unique state of catalyst materials emerging under specific reaction conditions and to establish catalyst structure-activity relationships. Recent studies highlight the importance of combining multiple operando techniques (multimodal approach) to gain complementary information as well as looking into chemical and material gradients and spatial variations on the reactor scale. In this work, we developed an operando UV–vis diffuse reflectance spectroscopy (DRS) setup compatible with a common fixed-bed tubular reactor. The design is based on optical calculations, validation experiments and signals considerations. A spatial resolution of 1 mm along the axial direction of the reactor was successfully demonstrated and combined with a time resolution of seconds with good signal to noise. CO oxidation over Pt/Al2O3 was performed as a proof of principle experiment demonstrating the capabilities of the new setup. The information gained by the space-resolved operando UV–vis DRS was combined with other space-resolved operando studies such as diffuse reflectance infrared Fourier transform spectroscopy (DRIFTS), gas sampling and temperature profiling. The study shows that the nature of active sites (Pt redox state) and thus the reaction mechanism alter with reaction temperature and also in space. Spatiotemporal UV–vis DRS is also demonstrated, showing the capability for transient studies with space-resolution.

Modulation of Neutrophil Activity by Soluble Complement Cleavage Products—An In-Depth Analysis

The cellular and fluid phase-innate immune responses of many diseases predominantly involve activated neutrophil granulocytes and complement factors. However, a comparative systematic analysis of the early impact of key soluble complement cleavage products, including anaphylatoxins, on neutrophil granulocyte function is lacking. Neutrophil activity was monitored by flow cytometry regarding cellular (electro-)physiology, cellular activity, and changes in the surface expression of activation markers. The study revealed no major effects induced by C3a or C4a on neutrophil functions. By contrast, exposure to C5a or C5a des-Arg stimulated neutrophil activity as reflected in changes in membrane potential, intracellular pH, glucose uptake, and cellular size. Similarly, C5a and C5a des-Arg but no other monitored complement cleavage product enhanced phagocytosis and reactive oxygen species generation. C5a and C5a des-Arg also altered the neutrophil surface expression of several complement receptors and neutrophil activation markers, including C5aR1, CD62L, CD10, and CD11b, among others. In addition, a detailed characterization of the C5a-induced effects was performed with a time resolution of seconds. The multiparametric response of neutrophils was further analyzed by a principal component analysis, revealing CD11b, CD10, and CD16 to be key surrogates of the C5a-induced effects. Overall, we provide a comprehensive insight into the very early interactions of neutrophil granulocytes with activated complement split products and the resulting neutrophil activity. The results provide a basis for a better and, importantly, time-resolved and multiparametric understanding of neutrophil-related (patho-)physiologies.

Mucin Transiently Sustains Coronavirus Infectivity through Heterogenous Changes in Phase Morphology of Evaporating Aerosol.

Respiratory pathogens can be spread though the transmission of aerosolised expiratory secretions in the form of droplets or particulates. Understanding the fundamental aerosol parameters that govern how such pathogens survive whilst airborne is essential to understanding and developing methods of restricting their dissemination. Pathogen viability measurements made using Controlled Electrodynamic Levitation and Extraction of Bioaerosol onto Substrate (CELEBS) in tandem with a comparative kinetics electrodynamic balance (CKEDB) measurements allow for a direct comparison between viral viability and evaporation kinetics of the aerosol with a time resolution of seconds. Here, we report the airborne survival of mouse hepatitis virus (MHV) and determine a comparable loss of infectivity in the aerosol phase to our previous observations of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Through the addition of clinically relevant concentrations of mucin to the bioaerosol, there is a transient mitigation of the loss of viral infectivity at 40% RH. Increased concentrations of mucin promoted heterogenous phase change during aerosol evaporation, characterised as the formation of inclusions within the host droplet. This research demonstrates the role of mucus in the aerosol phase and its influence on short-term airborne viral stability.

Tracing the composition of single e-cigarette aerosol droplets in situ by laser-trapping and Raman scattering

The partitioning of components between droplets and the gas phase in e-cigarette aerosols has a significant impact on deposition within the respiratory tract. However, exclusive detection of droplet composition has, so far, been elusive. Consequently, the dynamics of partitioning between droplets and the gas phase remains unknown. Here, we combine optical trapping of single droplets with in situ Raman scattering for destruction-free monitoring of e-cigarette droplet composition with a time resolution of seconds. We find that the initial droplet composition is very close to the composition of the e-liquid. Upon dilution with air, the droplet composition changes exponentially on a time scale of seconds, mainly because of evaporation of propylene glycol. The nicotine content in the droplet is controlled by the pH. Nicotine evaporates from the droplets under basic conditions, but remains in the liquid under acidic conditions. These results are crucial for advancing e-liquid research and manufacturing.

Evaluating the potential of hyperpolarised [1-13C] L-lactate as a neuroprotectant metabolic biosensor for stroke

Cerebral metabolism, which can be monitored by magnetic resonance spectroscopy (MRS), changes rapidly after brain ischaemic injury. Hyperpolarisation techniques boost 13C MRS sensitivity by several orders of magnitude, thereby enabling in vivo monitoring of biochemical transformations of hyperpolarised (HP) 13C-labelled precursors with a time resolution of seconds. The exogenous administration of the metabolite L-lactate was shown to decrease lesion size and ameliorate neurological outcome in preclinical studies in rodent stroke models, as well as influencing brain metabolism in clinical pilot studies of acute brain injury patients. The aim of this study was to demonstrate the feasibility of measuring HP [1-13C] L-lactate metabolism in real-time in the mouse brain after ischaemic stroke when administered after reperfusion at a therapeutic dose. We showed a rapid, time-after-reperfusion-dependent conversion of [1-13C] L-lactate to [1-13C] pyruvate and [13C] bicarbonate that brings new insights into the neuroprotection mechanism of L-lactate. Moreover, this study paves the way for the use of HP [1-13C] L-lactate as a sensitive molecular-imaging biosensor in ischaemic stroke patients after endovascular clot removal.

The ESRF dark-field x-ray microscope at ID06

We present an instrument for dark-field x-ray microscopy installed on beamline ID06 of the ESRF — the first of its kind. Dark-field x-ray microscopy uses full field illumination of the sample and provides three-dimensional (3D) mapping of micro-structure and lattice strain in crystalline matter. It is analogous to dark-field electron microscopy in that an objective lens magnifies diffracting features of the sample. The use of high-energy synchrotron x-rays, however, means that these features can be large and deeply embedded. 3D movies can be acquired with a time resolution of seconds to minutes. The field of view and spatial resolution can be adapted by simple reconfiguration of the x-ray objective lens, reaching spatial and angular resolution of 30-100 nm and 0.001°, respectively. The instrument furthermore allows pre-characterization of samples at larger length scales using 3DXRD or DCT, such that a region of interest (e.g. a single grain) can be selected for high-resolution studies without the need to dismount the sample. As examples of applications we show work on mapping the subgrains in plastically deformed iron and aluminum alloys, mapping domains and strain fields in ferroelectric crystals, and studies of biominerals. This ability to directly characterize complex, multi-scale phenomena in-situ is a key step towards formulating and validating multi-scale models that account for the entire heterogeneity of materials. As an outlook, we discuss future prospects for such multi-scale characterization by combining DFXM with 3DXRD/DCT, and coherent x-ray methods for coarser and finer length-scales, respectively.

Structure formation in organic thin films observed in real time by energy dispersive near-edge x-ray absorption fine-structure spectroscopy

We study the structure formation of 1,4,5,8-naphthalene-tetracarboxylicacid-dianhydride (NTCDA) multilayer films on Ag(111) surfaces by energy dispersive near-edge x-ray absorption fine-structure spectroscopy (NEXAFS) and photoelectron spectroscopy. The time resolution of seconds of the method allows us to identify several sub-processes, which occur during the post-growth three-dimensional structural ordering, as well as their characteristic time scales. After deposition at low temperature the NTCDA molecules are preferentially flat lying and the films exhibit no long-range order. Upon annealing the molecules flip into an upright orientation followed by an aggregation in a transient phase which exists for several minutes. Finally, three-dimensional islands are established with bulk-crystalline structure involving substantial mass transport on the surface and morphological roughening. By applying the Kolmogorov–Johnson–Mehl–Avrami model the activation energies of the temperature-driven sub-processes can be derived from the time evolution of the NEXAFS signal.

Kinetics of structural reorganizations in multilamellar photosynthetic membranes monitored by small-angle neutron scattering

We demonstrate the power of time-resolved small-angle neutron scattering experiments for the investigation of the structure and structural reorganizations of multilamellar photosynthetic membranes. In addition to briefly summarizing our results on thylakoid membranes isolated from higher plants and in unicellular organisms, we discuss the advantages and technical and methodological limitations of time-resolved SANS. We present a detailed and more systematical investigation of the kinetics of light-induced structural reorganizations in isolated spinach thylakoid membranes, which show how changes in the repeat distance and in the long-range order of the multilamellar membranes can be followed with a time resolution of seconds. We also present data from comparative measurements performed on thylakoid membranes isolated from tobacco.

Characterizing an Extractive Electrospray Ionization (EESI) Source for the Online Mass Spectrometry Analysis of Organic Aerosols

Organic compounds comprise a major fraction of tropospheric aerosol and understanding their chemical complexity is a key factor for determining their climate and health effects. We present and characterize here a new online technique for measuring the detailed chemical composition of organic aerosols, namely extractive electrospray ionization mass spectrometry (EESI-MS). Aerosol particles composed of soluble organic compounds were extracted into and ionized by a solvent electrospray, producing molecular ions from the aerosol with minimal fragmentation. We demonstrate here that the technique has a time resolution of seconds and is capable of making stable measurements over several hours. The ion signal in the MS was linearly correlated with the mass of aerosol delivered to the EESI source over the range tested (3-600 μg/m(3)) and was independent of particle size and liquid water content, suggesting that the entire particle bulk is extracted for analysis. Tandem MS measurements enabled detection of known analytes in the sub-μg/m(3) range. Proof-of-principle measurements of the ozonolysis of oleic acid aerosol (20 μg/m(3)) revealed the formation of a variety of oxidation products in good agreement with previous offline studies. This demonstrates the technique's potential for studying the product-resolved kinetics of aerosol-phase chemistry at a molecular level with high sensitivity and time resolution.

Three pools of zeaxanthin in Quercus coccifera leaves during light transitions with different roles in rapidly reversible photoprotective energy dissipation and photoprotection.

Under excess light, the efficient PSII light-harvesting antenna is switched into a photoprotected state in which potentially harmful absorbed energy is thermally dissipated. Changes occur rapidly and reversibly, enhanced by de-epoxidation of violaxanthin (V) to zeaxanthin (Z). This process is usually measured as non-photochemical quenching (NPQ) of chlorophyll (Chl) fluorescence. Using instrumentation for instantaneous leaf freezing, NPQ, spectral reflectance, and interconversions within the xanthophyll cycle with time resolution of seconds were recorded from Quercus coccifera leaves during low light (LL) to high light (HL) transitions, followed by relaxation at LL. During the first 30 s of both the LL to HL and HL to LL transitions, no activity of the xanthophyll cycle was detected, whereas 70–75% of the NPQ was formed and relaxed, respectively, by that time, the latter being traits of a rapidly reversible photoprotective energy dissipation. Three different Z pools were identified, which play different roles in energy dissipation and photoprotection. In conclusion, ΔpH was crucial to NPQ formation and relaxation in Q. coccifera during light transitions. Only a minor fraction of Z was associated to quenching, whereas the largest Z pool was not related to thermal dissipation. The latter is proposed to participate in photoprotection acting as antioxidant.

Time-resolved Measurements of Intracellular ATP in the Yeast Saccharomyces cerevisiae using a New Type of Nanobiosensor

Adenosine 5'-triphosphate is a universal molecule in all living cells, where it functions in bioenergetics and cell signaling. To understand how the concentration of ATP is regulated by cell metabolism and in turn how it regulates the activities of enzymes in the cell it would be beneficial if we could measure ATP concentration in the intact cell in real time. Using a novel aptamer-based ATP nanosensor, which can readily monitor intracellular ATP in eukaryotic cells with a time resolution of seconds, we have performed the first on-line measurements of the intracellular concentration of ATP in the yeast Saccharomyces cerevisiae. These ATP measurements show that the ATP concentration in the yeast cell is not stationary. In addition to an oscillating ATP concentration, we also observe that the concentration is high in the starved cells and starts to decrease when glycolysis is induced. The decrease in ATP concentration is shown to be caused by the activity of membrane-bound ATPases such as the mitochondrial F(0)F(1) ATPase-hydrolyzing ATP and the plasma membrane ATPase (PMA1). The activity of these two ATPases are under strict control by the glucose concentration in the cell. Finally, the measurements of intracellular ATP suggest that 2-deoxyglucose (2-DG) may have more complex function than just a catabolic block. Surprisingly, addition of 2-DG induces only a moderate decline in ATP. Furthermore, our results suggest that 2-DG may inhibit the activation of PMA1 after addition of glucose.

Fast time-resolved aerosol collector: proof of concept

Abstract. Atmospheric particles can be collected in the field on substrates for subsequent laboratory analysis via chemically sensitive single particle methods such as scanning electron microscopy with energy dispersive x-ray analysis. With moving substrates time resolution of seconds to minutes can be achieved. In this paper, we demonstrate how to increase the time resolution when collecting particles on a substrate to a few milliseconds to provide real-time information. Our fast time-resolved aerosol collector ("Fast-TRAC") microscopically observes the particle collection on a substrate and records an on-line video. Particle arrivals are resolved to within a single frame (4–17 ms in this setup), and the spatial locations are matched to the subsequent single particle analysis. This approach also provides in-situ information on particle size and number concentration. Applications are expected in airborne studies of cloud microstructure, pollution plumes, and surface long-term monitoring.

Time-lapse imaging of dendritic spines in vitro

Dendritic spines are small protrusions present postsynaptically at approximately 90% of excitatory synapses in the brain. Spines undergo rapid spontaneous changes in shape that are thought to be important for alterations in synaptic connectivity underlying learning and memory. Visualization of these dynamic changes in spine morphology are especially challenging because of the small size of spines (approximately 1 microm). Here we describe a microscope system, based on a spinning-disk confocal microscope, suitable for imaging mature dendritic spines in brain slice preparations, with a time resolution of seconds. We discuss two commonly used in vitro brain slice preparations and methods for transfecting them. Preparation and transfection require approximately 1 d, after which slices must be cultured for at least 21 d to obtain spines of mature morphology. We also describe imaging and computer analysis routines for studying spine motility. These procedures require in the order of 2 to 4 h.

Coupling between Clathrin-Coated-Pit Invagination, Cortactin Recruitment, and Membrane Scission Observed in Live Cells

During clathrin-mediated endocytosis, membrane scission marks the isolation of a cargo-laden clathrin-coated pit (CCP) from the cell exterior. Here we used live-cell imaging of a pH-sensitive cargo to visualize the formation of clathrin-coated vesicles (CCVs) at single CCPs with a time resolution of seconds. We show that CCPs are highly dynamic and can produce multiple vesicles in succession. Using alternating evanescent field and epifluorescence illumination, we show that CCP invagination and scission are tightly coupled, with scission coinciding with maximal displacement of CCPs from the plasma membrane and with peak recruitment of cortactin-DsRed, a dynamin and F-actin binding protein. Finally, perturbing actin polymerization with latrunculin-B drastically reduces the efficiency of membrane scission and affects many aspects of CCP dynamics. We propose that CCP invagination, actin polymerization, and CCV formation are highly coordinated for efficient endocytosis.

Automatic monitoring of mechano-electrical transduction in the guinea pig cochlea

We have estimated the transfer curve relating instantaneous sound pressure in the ear canal to instantaneous receptor current through the outer hair cells (OHCs) in the basal turn of the guinea pig cochlea using the cochlear microphonic (CM) elicited by continuous 200 Hz tones. The transfer curve is well approximated by a Boltzmann activation curve which has been automatically analysed using a custom-built electronic circuit which continuously derives the three parameters defining the curve with a time resolution of seconds. This technique offers a convenient method of monitoring changes in OHC mechano-electrical transduction due to cochlear disturbances, and allows the investigation of cochlear homeostasis over the course of hours. We present here details of the technique, evidence that the recordings are minimally contaminated by neural responses, and normative data on the changes in the parameters with sound level. As the level of the 200 Hz tone increases, the equivalent operating point on the transfer curve migrates in a way consistent with a movement of the organ of Corti towards scala tympani or a contraction of the outer hair cells. Surprisingly, the effective slope of the curve which represents the mechanical sensitivity of the transduction process decreases over an 8 to 1 range as the level of the 200 Hz tone is increased. The effect of this variation is that the amplitude of the equivalent mechanical displacement input to the mechano-electrical transduction process appears to increase by a mere 2 to 1 while the sound level increases by a factor of 20 to 1. These changes are not neurally mediated, since they also occur in the presence of tetrodotoxin and the blocker of afferent neurotransmission, kainate.

Initial Activity of Reduced Chromia/Alumina Catalyst inn-Butane Dehydrogenation Monitored by On-Line FT–IR Gas Analysis

The initial activity of chromia/alumina catalyst (13 wt% Cr) inn-butane dehydrogenation was studied in a flow reactor at 853 K. The initial activity was determined by on-line FT–IR gas analysis, which enabled sampling of the gaseous product mixture at a time resolution of seconds. The catalysts were processed in repeated cycles of oxidation, reduction, and dehydrogenation usingn-butane, methane, hydrogen, or carbon monoxide as reducing agents. Withn-butane, methane, and hydrogen the dehydrogenation activity was associated with Cr3+species apparently formed in the reduction of high-valence Cr species. The catalyst reduced with carbon monoxide at 853 K showed poor initial selectivity for butenes and, relative to the other catalysts, stronger formation of coke in dehydrogenation. The initial selectivity for butenes increased with time on stream, however, and within 2 min reached the level obtained with the other catalysts. Simultaneous data relating the initial activity, coke content, and some of the physicochemical properties of the catalyst indicated that the surfaces of all catalysts were modified to some extent by the successive reaction cycles.

FT-IR in the Quantitative Analysis of Gaseous Hydrocarbon Mixtures

An FT-IR spectrometer provided with software for multi component analysis of FR spectra was used for simultaneous determination of up to 12 hydrocarbons in a gaseous mixture. The hydrocarbons, ranging from methane to n-butane, were selected to simulate reaction product mixtures obtained in the dehydrogenation of n-butane. The concentrations of the hydrocarbons in the sample were determined by fitting to the sample spectrum a set of pure calibration spectra of the gases present in the sample. In principle, the entire wavenumber range of the low-resolution IR spectrum could be utilized in the fit. The reliability of the results was checked from the residual spectrum, obtained by subtracting the fitted from the measured spectrum. The residual spetrum will be pure noise when the set of calibration spectra chosen for the analysis fits exactly with the sample spectrum. Precision of the results, expressed as average relative standard deviation, was ±4%. Concentrations of most of the hydrocarbons were determined with an error less than ±10%. The concentrations of isobutane, trans-2-butene, propane, and ethane deviated by more than 10% from the reference concentrations. The sum concentration of ethane and propane, however, deviated less than ±1% from the reference. Difficulties in determining ethane and propane accurately were due to the broad nature and small number of peaks in their IR spectra. IR spectrometry with multicomponent analysis of spectra provides a rapid and powerful method with potential for the study of catalytic reactions at a time resolution of seconds

Interpretation of Oil-Water Relative Permeabilities from Centrifuge Experiments

ABSTRACT Collection of production data from centrifuge displacement with a time resolution of seconds and volume resolution of 4 µl provides the precision for interpreting the transient data for the displaced phase relative permeability curve. Interpretation for the relative permeability requires a simulation model that includes all relevant phenomena that affects the displacement. This includes the variation of the centrifugal force within the sample and during the centrifuge start-up period. Capillary pressure is a significant factor in both the initiation and cessation of production and thus must also be included in the analysis. The finite mobility of the invading phase places limitations on the portion of the relative permeability curve that is in the region of the shock. In addition, the finite mobility of the invading phase reduces the sensitivity to the initial (end ppinf) relative permeability of the displaced phase. The design of experiments in mixed wet systems require a balance between the Brownell-Katz (or Bond) number criteria for displacement of a disconnected phase and retention due to a capillary end effect.