One of the key challenges in the field of nanoparticle (NP) analysis is in producing reliable and reproducible characterisation data for nanomaterials. This study looks at the reproducibility using a relatively new, but rapidly adopted, technique, Nanoparticle Tracking Analysis (NTA) on a range of particle sizes and materials in several different media. It describes the protocol development and presents both the data and analysis of results obtained from 12 laboratories, mostly based in Europe, who are primarily QualityNano members. QualityNano is an EU FP7 funded Research Infrastructure that integrates 28 European analytical and experimental facilities in nanotechnology, medicine and natural sciences with the goal of developing and implementing best practice and quality in all aspects of nanosafety assessment. This study looks at both the development of the protocol and how this leads to highly reproducible results amongst participants. In this study, the parameter being measured is the modal particle size.

Red blood cell (RBC) parameters such as morphology, volume, refractive index, and hemoglobin content are of great importance for diagnostic purposes. Existing approaches require complicated calibration procedures and robust cell perturbation. As a result, reference values for normal RBC differ depending on the method used. We present a way for measuring parameters of intact individual RBCs by using digital holographic microscopy (DHM), a new interferometric and label-free technique with nanometric axial sensitivity. The results are compared with values achieved by conventional techniques for RBC of the same donor and previously published figures. A DHM equipped with a laser diode (k 5 663 nm) was used to record holograms in an off-axis geometry. Measurements of both RBC refractive indices and volumes were achieved via monitoring the quantitative phase map of RBC by means of a sequential perfusion of two isotonic solutions with different refractive indices obtained by the use of Nycodenz (decoupling procedure). Volume of RBCs labeled by membrane dye Dil was analyzed by confocal microscopy. The mean cell volume (MCV), red blood cell distribution width (RDW), and mean cell hemoglobin concentration (MCHC) were also measured with an impedance volume analyzer. DHM yielded RBC refractive index n 5 1.418 AE 0.012, volume 83 AE 14 fl, MCH 5 29.9 pg, and MCHC 362 AE 40 g/l. Erythrocyte MCV, MCH, and MCHC achieved by an impedance volume analyzer were 82 fl, 28.6 pg, and 349 g/l, respectively. Confocal microscopy yielded 91 AE 17 fl for RBC volume. In conclusion, DHM in combination with a decoupling procedure allows measuring noninvasively volume, refractive index, and hemoglobin content of single-living RBCs with a high accuracy. ' 2008 International Society for Advancement of Cytometry Key terms digital holographic microscopy; laser scanning confocal microscopy; impedance volume analyzer; red blood cell volume; MCV; refractive index; mean corpuscular hemoglobin concentration; MCHC

Cell membrane fluctuations (CMF) of human erythrocytes, measured by point dark field microscopy, were shown to depend, to a large extent, on intracellular MgATP (Levin, S.V., and R. Korenstein. 1991. Biophys. J. 60:733–737). The present study extends that investigation and associates CMF with F-actin's ATPase activity. MgATP was found to reconstitute CMF in red blood cell (RBC) ghosts and RBC skeletons to their levels in intact RBCs, with an apparent K d of 0.29 mM. However, neither non-hydrolyzable ATP analogues (AMP-PNP, ATPγS) nor hydrolyzable ones (ITP, GTP), were able to elevate CMF levels. The inhibition of ATPase activity associated with the RBC's skeleton, carried out either by the omission of the MgATP substrate or by the use of several inhibitors (vanadate, phalloidin, and DNase I), resulted in a strong decrease of CMF. We suggest that the actin's ATPase, located at the pointed end of the short actin filament, is responsible for the MgATP stimulation of CMF in RBCs.

Photochemical studies of the effects of temperature, pH, and dehydration on the formation and back photoreaction of the M412 intermediate in the photocycle of light-adapted bacteriorhodopsin (bR570) are carried out. Continuous illumination experiments in the range between -40 and -90 degrees C indicate that at low temperatures branching occurs at the stage of the L550 intermediate in which a back reaction to the parent pigment competes with the formation of M412. At low temperatures the yield of M412 is markedly increased at high pH. The effect is attributed to the catalytic action of a protein group of pK congruent to 10 on the rate of the L550 leads to M412 process. Our results, taken together with previous evidence for deprotonation of a tyrosine during the L550 leads to M412 transition, suggest that the formation of a tyrosinate ion is a prerequisite for deprotonation of the Schiff base. A model is proposed in which both the Schiff base and the tyrosine translocate their protons to two acceptor groups, A1 and A2, accessible to the outside of the cell through a segment of a proton wire. The model accounts for the observation that up to two photons may be pumped per cycle. The proton-pump mechanism is analyzed in terms of a generalized kinetic scheme for pumping. In contrast to current models for proton pumping which are based on a (primary) light-induced accessibility change of the chromophore (class I models), we introduce a new class (II) of models based exclusively on pK changes. We suggest that in bR570 the Schiff base and the tyrosine are accessible to protons on the outside surface of the membrane. An analysis of the back photoreaction from M412 tends to favor class II models over previous class I models.

Extracellular f luid macroviscosity (EFM), modified by macromolecular cosolvents as occurs in body f luids, has been shown to affect cell membrane protein activities but not isolated proteins. In search for the mechanism of this phenomenon, we examined the effect of EFM on mechanical f luctuations of the cell membrane of human erythrocytes. The macroviscosity of the external medium was varied by adding to it various macromolecules [dextrans (70, 500, and 2,000 kDa), polyethylene glycol (20 kDa), and carboxymethyl-cellulose (100 kDa)], which differ in size, chemical nature, and in their capacity to increase f luid viscosity. The parameters of cell membrane f luctuations (maximal amplitude and half-width of amplitude distribution) were diminished with the elevation of solvent macroviscosity, regardless of the cosolvent used to increase EFM. Because thermally driven membrane f luctuations cannot be damped by elevation of EFM, the existence of a metabolic driving force is suggested. This is supported by the finding that in ATPdepleted red blood cells elevation of EMF did not affect cell membrane f luctuations. This study demonstrates that (i) EFM is a regulator of membrane dynamics, providing a possible mechanism by which EFM affects cell membrane activities; and (ii) cell membrane f luctuations are driven by a metabolic driving force in addition to the thermal one.The viscosity of body fluids is determined by the level of macromolecules consisting of proteins, lipoproteins, and polysacharides (1). Accordingly, elevated plasma viscosity has been observed in various diseases associated with increased levels of proteins and lipoproteins, such as diabetes, hyperlipidemia, macroglobulinemia, multiple myeloma, nephrosis, and others (1-5). Various studies have shown that solvent viscosity affects protein dynamics and reactions (6-10). However, in these studies the solvent viscosity was modified by the addition of high concentrations of small cosolvents such as glycerol and sucrose, producing relatively high viscosity levels. This is incompatible with physiological and pathological states, where fluid viscosity is altered by small concentrations of large macromolecules (1). Other studies, in which the viscosity was elevated by macromolecular cosolvents, have shown that extracellular fluid macroviscosity (EFM) is a regulator of cellular processes, such as secretion of renin (11) and lipoproteins (12), phospholipase A 2 activity at the cell membrane (13, 14), and ganglioside metabolism (15). In search of the mechanism of this phenomenon, the effect of macroviscosity, as modified by macromolecules, on isolated proteins in aqueous solutions was examined (16,17). It was found that the effect of solvent viscosity decreases with increasing molecular weight of the cosolvent and is practically diminished when the cosolvent molecular weight exceeds that of the protein. Because the activity of cell membrane enzymes is known to be sensitive to the physical properties of the membrane (18), we considered the possibility that the...