Unravelling the fluorescence kinetics of light-harvesting proteins with simulated measurements.

The plant light-harvesting pigment-protein complex LHCII is the major antenna sub-unit of PSII and is generally (though not universally) accepted to play a role in photoprotective energy dissipation under high light conditions, a process known Non-Photochemical Quenching (NPQ). The underlying mechanisms of energy trapping and dissipation within LHCII are still debated. Various models have been proposed for the underlying molecular detail of NPQ, but they are often based on different interpretations of very similar transient absorption measurements of isolated complexes. Here we present a simulated measurement of the fluorescence decay kinetics of quenched LHCII aggregates to determine whether this relatively simple measurement can discriminate between different potential NPQ mechanisms. We simulate not just the underlying physics (excitation, energy migration, quenching and singlet-singlet annihilation) but also the signal detection and typical experimental data analysis. Comparing this to a selection of published fluorescence decay kinetics we find that: (1) Different proposed quenching mechanisms produce noticeably different fluorescence kinetics even at low (annihilation free) excitation density, though the degree of difference is dependent on pulse width. (2) Measured decay kinetics are consistent with most LHCII trimers becoming relatively slow excitation quenchers. A small sub-population of very fast quenchers produces kinetics which do not resemble any observed measurement. (3) It is necessary to consider at least two distinct quenching mechanisms in order to accurately reproduce experimental kinetics, supporting the idea that NPQ is not a simple binary switch.

Observation of Aerosol Generation by Human Subjects During Cardiopulmonary Exercise Testing Using a High-Powered Laser Technique: A Pilot Project.

Purpose: Human respiratory aerosols may have important implications for transmission of pathogens. The study of aerosol production during vigorous breathing activities such as exercise is limited. In particular, data on aerosol production during cardiopulmonary exercise testing (CPET) are lacking. Methods: In this pilot project, we used a high-powered, pulsed Nd:YAG laser to illuminate a region of interest in front of two healthy adult subjects during CPET. Subjects exercised to the point of respiratory compensation. Images were captured with a high-speed, high-resolution camera to determine net exhaled particle (NEP) counts at different phases of CPET, including resting breathing, submaximal exercise, peak exercise, and active recovery. Experiments were performed with the room ventilation activated. Results: Net exhaled particle counts remained relatively constant until late/peak exercise when they decreased prior to rebounding into recovery. NEP counts at resting breathing were higher than those reported using other methods of measurement. Exhaled particles were in the submicron size range. Conclusion: Our method of aerosol particle quantification enables measurement of significant quantities of ultrafine particles and dynamic assessment of aerosol production during CPET. The unique pattern of aerosol production observed during submaximal and peak exercise suggests that extension of results from resting breathing to CPET may not be appropriate.

Trivial Excitation Energy Transfer to Carotenoids Is an Unlikely Mechanism for Non-photochemical Quenching in LHCII.

Higher plants defend themselves from bursts of intense light via the mechanism of Non-Photochemical Quenching (NPQ). It involves the Photosystem II (PSII) antenna protein (LHCII) adopting a conformation that favors excitation quenching. In recent years several structural models have suggested that quenching proceeds via energy transfer to the optically forbidden and short-lived S 1 states of a carotenoid. It was proposed that this pathway was controlled by subtle changes in the relative orientation of a small number of pigments. However, quantum chemical calculations of S 1 properties are not trivial and therefore its energy, oscillator strength and lifetime are treated as rather loose parameters. Moreover, the models were based either on a single LHCII crystal structure or Molecular Dynamics (MD) trajectories about a single minimum. Here we try and address these limitations by parameterizing the vibronic structure and relaxation dynamics of lutein in terms of observable quantities, namely its linear absorption (LA), transient absorption (TA) and two-photon excitation (TPE) spectra. We also analyze a number of minima taken from an exhaustive meta-dynamical search of the LHCII free energy surface. We show that trivial, Coulomb-mediated energy transfer to S 1 is an unlikely quenching mechanism, with pigment movements insufficiently pronounced to switch the system between quenched and unquenched states. Modulation of S 1 energy level as a quenching switch is similarly unlikely. Moreover, the quenching predicted by previous models is possibly an artifact of quantum chemical over-estimation of S 1 oscillator strength and the real mechanism likely involves short-range interaction and/or non-trivial inter-molecular states.

Milk-alkali syndrome: a 'quick ease' or a 'long-lasting problem'.

SUMMARY: We report the case of a 65-year-old female who presented with symptomatic hypercalcaemia (corrected calcium of 4.57 mmol/L) with confusion, myalgias and abdominal discomfort. She had a concomitant metabolic alkalosis (pH 7.46, HCO3- 40 mmol/L, pCO2 54.6 mmHg). A history of significant Quick-Eze use (a calcium carbonate based antacid) for abdominal discomfort, for 2 weeks prior to presentation, suggested a diagnosis of milk-alkali syndrome (MAS). Further investigations did not demonstrate malignancy or primary hyperparathyroidism. Following management with i.v. fluid rehydration and a single dose of i.v. bisphosphonate, she developed symptomatic hypocalcaemia requiring oral and parenteral calcium replacement. She was discharged from the hospital with stable biochemistry on follow-up. This case demonstrates the importance of a detailed history in the diagnosis of severe hypercalcaemia, with MAS representing the third most common cause of hypercalcaemia. We discuss its pathophysiology and clinical importance, which can often present with severe hypercalcaemia that can respond precipitously to calcium-lowering therapy. LEARNING POINTS: Milk-alkali syndrome is an often unrecognised cause for hypercalcaemia, but is the third most common cause of admission for hypercalcaemia. Calcium ingestion leading to MAS can occur at intakes as low as 1.0-1.5 g per day in those with risk factors. Early recognition of this syndrome can avoid the use of calcium-lowering therapy such as bisphosphonates which can precipitate hypocalcaemia.

Ice Scallops: A Laboratory Investigation of the Ice-Water Interface.

Ice scallops are a small-scale (5-20cm) quasi-periodic ripple pattern that occurs at the ice-water interface. Previous work has suggested that scallops form due to a self-reinforcing interaction between an evolving ice-surface geometry, an adjacent turbulent flow field, and the resulting differential melt rates that occur along the interface. In this study, we perform a series of laboratory experiments in a refrigerated flume to quantitatively investigate the mechanisms of scallop formation and evolution in high resolution. Using particle-image velocimetry, we probe an evolving ice-water boundary layer at sub-millimeter scales and 15Hz frequency. Our data reveals three distinct regimes of ice-water interface evolution: A transition from flat to scalloped ice; an equilibrium scallop geometry; and an adjusting scallop interface. We find that scalloped ice geometry produces a clear modification to the ice-water boundary layer, characterized by a time-mean recirculating eddy feature that forms in the scallop trough. Our primary finding is that scallops form due to a self reinforcing feedback between the ice-interface geometry and shear production of turbulent kinetic energy in the flow interior. The length of this shear production zone is therefore hypothesized to set the scallop wavelength.

In situ visualization and characterization of aerosol droplets in an inductively coupled plasma.

Laser-scattering techniques are utilized for the first time to visualize the aerosol droplets in an inductively coupled plasma (ICP) torch from the nebulizer tip to the site of analytical measurements. The resulting images provide key information about the spatial distribution of the aerosol introduced by direct injection and conventional sample introduction devices: (1) a direct injection high-efficiency nebulizer (DIHEN); (2) a large-bore DIHEN; and (3) a MicroFlow PFA nebulizer with a PFA Scott-type spray chamber. Moreover, particle image velocimetry is used to study the in situ behavior of the aerosol before interaction with the plasma, while the individual surviving droplets are explored by particle tracking velocimetry. Directly introduced aerosols are highly scattered across the plasma torch as a result of their radial motion, indicating less than optimum sample consumption efficiency for the current direct injection devices. Further, the velocity distribution of the surviving droplets demonstrates the importance of the initial droplet velocities in complete desolvation of the aerosol for optimum analytical performance in ICP spectrometries. These new observations are critical in the design of the next-generation direct injection devices for lower sample consumption, higher sensitivity, lower noise levels, suppressed matrix effects, and developing smart spectrometers.

Spatial mapping of droplet velocity and size for direct and indirect nebulization in plasma spectrometry.

Two novel laser-based imaging techniques centered on particle image velocimetry and optical patternation are used to map and contrast the size and velocity distributions for indirect and direct pneumatic nebulizations in plasma spectrometry. The flow field of droplets is illuminated by two pulses from a thin laser sheet with a known time difference. The scattering of the laser light from droplets is captured by a charge-coupled device (CCD), providing two instantaneous images of the particles. Pointwise cross-correlation of the corresponding images yields a two-dimensional velocity map of the aerosol velocity field. For droplet size distribution studies, the solution is doped with a fluorescent dye and both laser-induced florescence (LIF) and Mie scattering images are captured simultaneously by two CCDs with the same field of view. The ratio of the LIF/Mie images provides relative droplet size information, which is then scaled by a point calibration method via a phase Doppler particle analyzer. Two major findings are realized for three nebulization systems: (1) a direct injection high-efficiency nebulizer (DIHEN); (2) a large-bore DIHEN; and (3) a PFA microflow nebulizer with a PFA Scott-type spray chamber. First, the central region of the aerosol cone from the direct injection nebulizers and the nebulizer-spray chamber arrangement consists of fast (>13 and >8 m/s, respectively) and fine (<10 and <5 microm, respectively) droplets as compared to slow (<4 m/s) and large (>25 microm) droplets in the fringes. Second, the spray chamber acts as a momentum separator, rather than a droplet size selector, as it removes droplets having larger sizes or velocities. The concepts and results presented in this research may be used to develop smart-tunable nebulizers, for example, by using the measured momentum as a feedback control for adjusting the nebulizer, i.e., its operating conditions, its critical dimensions, or both.