Continuous suspension cell culture monitoring in bioreactors using quantitative imaging

Monitoring of suspension cell cultures often relies on sampling followed by a staining procedure. Estimations of cell count and cell viability are traditionally performed once a day using Trypan-Blue cell exclusion as a method of choice. Stained samples are destroyed afterwards creating toxic waste. Sampling a bioreactor and counting cells involve manual operations and weekend work is regularly needed. Differential Digital Holographic Microscopy (DDHM) is a new quantitative imaging technique that allows cell counting as well as cell viability monitoring in a continuous, label-free set-up. No need for sampling (thus eliminating the risk of contamination), staining and waiting for results generated by an off-line counter: results are available in nearly real-time during the whole run. Compared to classical light microscopy, Differential Digital Holographic Microscopy offers: The ability to refocus images post acquisition The collection of quantitative phase information (optical density), covering the shape and density of an object. This quantitative phase parameter (not captured by the human eye) is the key advantage in numerous applications developed at OVIZIO. DDHM helps the operator to continuously track total cell density and cell viability, while the OsOne software plots the cell growth curve, live on the screen. Moreover, OsOne also shows real-time images of cells, offering the experienced operator a particularly convenient tool to check the condition of the cell culture. In this study, we compared the results generated by the iLine F microscope with off-lines methods applying sampling and Trypan-Blue staining. OVIZIO’s iLine F was benchmarked versus the Vi-Cell XR (Beckman Coulter). A bioreactor equipped with a BioConnect (OVIZIO’s continuous, closed loop, sampling device) plugged into an iLine F was inoculated with CHO cells at 0.3x106 viable cells/mL in CD-CHO medium (Life Technologies) for a final volume of 2L. The culture was sampled daily via the usual sampling port for Vi-Cell cell count. The iLine F was set to generate 2 data points (cell counts and viability measurement) per hour. The culture was left to grow in batch mode so it was possible to also capture the decrease in cell viability at the end of the bioreactor run. An excellent correlation factor R² was obtained for the viable cell density demonstrating that the results achieved with the label-free DDHM method are in line with current methods applying Trypan-Blue staining. Furthermore, the iLine F shows the benefit of having the full trend of the culture which can be more relevant than a single point, on a single sample, once a day. The availability of full data at the single cell level, for the whole experiment, allows to envision the use of the iLine F in a PAT approach. Indeed the large amount of data produced can be used to perform various statistical analysis on the cell population in order to define and control critical parameters of the cell culture process

Phase transition close to room temperature in BiFeO3 thin films

BiFeO3 (BFO) multiferroic oxide has a complex phase diagram that can be mapped by appropriately substrate-induced strain in epitaxial films. By using Raman spectroscopy, we conclusively show that films of the so-called supertetragonal T-BFO phase, stabilized under compressive strain, displays a reversible temperature-induced phase transition at about 100\circ, thus close to room temperature.Comment: accepted in J. Phys.: Condens. Matter (Fast Track Communication

Analysis of segmental residual growth after progressive bone lengthening in congenital lower limb deformity

SummaryIntroductionThe issue of prognosis in limb length discrepancy in children affected by congenital abnormality remains a subject of concern. Therapeutic strategy must take length prediction into account, to adapt equalization techniques and the timing of treatment. Initial prognosis, however, may need revising after completion of one or several surgical interventions on the pathologic limb. The aim of this study was to determine the different types of growth response that a bone segment can present after progressive lengthening in case of congenital limb length discrepancy.Materials and methodsA series of 114 bone lengthenings with external fixator, performed in 36 girls and 50 boys with congenital lower limb length discrepancy, was retrospectively analyzed. Bone segment growth rates were measured before lengthening, during the first year after frame removal and finally over long-term follow-up, calculating the ratios of radiological bone length to the number of months between two measurements. Mean follow-up was 4.54±0.2 years.ResultsChanges in short- and long-term growth rate distinguished five patterns of bone behavior after lengthening, ranging from growth acceleration to total inhibition.DiscussionThese five residual growth patterns depended on certain factors causing acceleration or, on the contrary, slowing down of growth: age at the lengthening operation, percentage lengthening, and minimal period between two lengthenings. These criteria help optimize conditions for resumed growth after progressive segmental lengthening, avoiding conditions liable to induce slowing down or inhibition, and providing a planning aid in multi-step lengthening programs.Level of evidenceLevel IV. Retrospective study

Infrared and THz studies of polar phonons and improper magnetodielectric effect in multiferroic BFO3 ceramics

BFO3 ceramics were investigated by means of infrared reflectivity and time domain THz transmission spectroscopy at temperatures 20 - 950 K, and the magnetodielectric effect was studied at 10 - 300 K, with the magnetic field up to 9 T. Below 175 K, the sum of polar phonon contributions into the permittivity corresponds to the value of measured permittivity below 1 MHz. At higher temperatures, a giant low-frequency permittivity was observed, obviously due to the enhanced conductivity and possible Maxwell-Wagner contribution. Above 200 K the observed magnetodielectric effect is caused essentially through the combination of magnetoresistance and the Maxwell-Wagner effect, as recently predicted by Catalan (Appl. Phys. Lett. 88, 102902 (2006)). Since the magnetodielectric effect does not occur due to a coupling of polarization and magnetization as expected in magnetoferroelectrics, we call it improper magnetodielectric effect. Below 175 K the magnetodielectric effect is by several orders of magnitude lower due to the decreased conductivity. Several phonons exhibit gradual softening with increasing temperature, which explains the previously observed high-frequency permittivity increase on heating. The observed non-complete phonon softening seems to be the consequence of the first-order nature of the ferroelectric transition.Comment: subm. to PRB. revised version according to referees' report

DNA immunisation. New histochemical and morphometric data.

Splenic germinal center reactions were measured during primary response to a plasmidic DNA intramuscular injection. Cardiotoxin-pretreated Balb/c mice were immunized with DNA plasmids encodmg or not the SAG1 protein, a membrane antigen of Toxoplasma gondii. Specific anti-SAG1 antibodies were detected on days 16 and 36 after injection of coding plasmids. The results of ELISAs showed that the SAG1-specific antibodies are of the IgG2a class. Morphometric analyses were done on serial immunostained cryosections of spleen and draining or non-draining lymph nodes. This new approach made it possible to evaluate the chronological changes induced by DNA immunisation in the germinal centres (in number and in size). Significant increases in the number of germinal centres were measured in the spleen and only in draining lymph nodes after plasmid injection, the measured changes of the germinal centers appeared to result from the adjuvant stimulatory effect of the plasmidic DNA since both the coding and the noncoding plasmid DNA induced them. No measurable changes were recorded in the T-dependent zone of lymph organs

Voxelization of Free-Form Solids Represented by Catmull-Clark Subdivision Surfaces

www.cs.uky.edu/∼cheng Abstract. A voxelization technique and its applications for objects with arbitrary topology are presented. It converts a free-form object from its continuous geometric representation into a set of voxels that best approximates the geometry of the object. Unlike traditional 3D scan-conversion based methods, our voxelization method is performed by recursively subdividing the 2D parameter space and sampling 3D points from selected 2D parameter space points. Moreover, our voxelization of 3D closed objects is guaranteed to be leak-free when a 3D flooding operation is performed. This is ensured by proving that our voxelization results satisfy the properties of separability, accuracy and minimality.

Magnetoelectric ordering of BiFeO3 from the perspective of crystal chemistry

In this paper we examine the role of crystal chemistry factors in creating conditions for formation of magnetoelectric ordering in BiFeO3. It is generally accepted that the main reason of the ferroelectric distortion in BiFeO3 is concerned with a stereochemical activity of the Bi lone pair. However, the lone pair is stereochemically active in the paraelectric orthorhombic beta-phase as well. We demonstrate that a crucial role in emerging of phase transitions of the metal-insulator, paraelectric-ferroelectric and magnetic disorder-order types belongs to the change of the degree of the lone pair stereochemical activity - its consecutive increase with the temperature decrease. Using the structural data, we calculated the sign and strength of magnetic couplings in BiFeO3 in the range from 945 C down to 25 C and found the couplings, which undergo the antiferromagnetic-ferromagnetic transition with the temperature decrease and give rise to the antiferromagnetic ordering and its delay in regard to temperature, as compared to the ferroelectric ordering. We discuss the reasons of emerging of the spatially modulated spin structure and its suppression by doping with La3+.Comment: 18 pages, 5 figures, 3 table

Interaction of low-energy electrons with surface polarity near ferroelastic domain boundaries

We derive surface polarity at and near ferroelastic domain boundaries from molecular dynamics simulations based on an ionic spring model. Interatomic gradient forces lead to flexoelectricity which, in turn, generates polarity at the surface and in twin boundaries. We then derive generic properties of electron scattering spectra equivalent to those observed in low-energy electron microscopy (LEEM) and mirror electron microscopy (MEM) experiments. Negatively (positively) charged surfaces reflect (attract) incident electrons with low kinetic energy. The electron images reveal the valley and ridge surface structures near the intersection of the twin boundary and the surface. Polarity in surface layers is predicted to be visible in LEEM and MEM spectra at neutral surfaces, but much less when surfaces are charged. Inward polarity reflects electrons similar to negative surface charges, and outward polarity backscatters electrons like positive surface charges. Both the polarity in the twin boundary and the physical topography scatter electrons, consistent with experimental LEEM and MEM experiments on CaTi O 3 with (001) and (111) surface terminations.EPSR

Control of surface potential at polar domain walls in a nonpolar oxide

Ferroic domain walls could play an important role in microelectronics, given their nanometric size and often distinct functional properties. Until now, devices and device concepts were mostly based on mobile domain walls in ferromagnetic and ferroelectric materials. A less explored path is to make use of polar domain walls in nonpolar ferroelastic materials. Indeed, while the polar character of ferroelastic domain walls has been demonstrated, polarization control has been elusive. Here, we report evidence for the electrostatic signature of the domain-wall polarization in nonpolar calcium titanate (CaTiO3). Macroscopic mechanical resonances excited by an ac electric field are observed as a signature of a piezoelectric response caused by polar walls. On the microscopic scale, the polarization in domain walls modifies the local surface potential of the sample. Through imaging of surface potential variations, we show that the potential at the domain wall can be controlled by electron injection. This could enable devices based on nondestructive information readout of surface potential