Time-lapse Optical Microscopy Research Articles

Puzzle-shaped cells and the mechanical response of tobacco (Nicotiana tabacum L.) seed coats

Abstract The seed coat of tobacco displays an intriguing cellular pattern characterised by puzzle-like shapes whose specific function is unknown. Here, we perform a detailed investigation of the structure of tobacco seeds by electron microscopy and then follow the germination process by time lapse optical microscopy. We use particle image velocimetry to reveal the local deformation fields and perform compression experiments to study the mechanical properties of the seeds as a function of their hydration. To understand the mechanical role of the observed coat structure, we perform finite element calculations comparing structure with puzzle-shaped cells with similar structures lacking re-entrant features. The results indicate that puzzle-shaped cells act as stress suppressors and reduce the Poisson’s ratio of the seed coat structure. We thus conclude that the peculiar cellular structure of these seed coats serves a mechanical purpose that could be relevant to control germination.

Understanding the diagnosis of catheter-related bloodstream infection: real-time monitoring of biofilm growth dynamics using time-lapse optical microscopy.

The differential time to positivity (DTTP) technique is recommended for the conservative diagnosis of catheter-related bloodstream infection (C-RBSI). The technique is based on a 120-minute difference between microbial growth in blood drawn through the catheter and blood drawn through a peripheral vein. However, this cut-off has failed to confirm C-RBSI caused by Candida spp. and Staphylococcus aureus. We hypothesized that the biofilm of both microorganisms disperses faster than that of other microorganisms and that microbial load is rapidly equalized between catheter and peripheral blood. Therefore, our aim was to compare the biofilm dynamics of various microorganisms. Biofilm of ATCC strains of methicillin-resistant Staphylococcus epidermidis, methicillin-susceptible S. aureus, Enterococcus faecalis, Escherichia coli and Candida albicans was grown on silicon disks and analyzed using time-lapse optical microscopy. The time-lapse images of biofilms were processed using ImageJ2 software. Cell dispersal time and biofilm thickness were calculated. The mean (standard deviation) dispersal time in C. albicans and S. aureus biofilms was at least nearly 3 hours lower than in biofilm of S. epidermidis, and at least 15 minutes than in E. faecalis and E. coli biofilms. Our findings could explain why early dissemination of cells in C. albicans and S. aureus prevents us from confirming or ruling out the catheter as the source of the bloodstream infection using the cut-off of 120 minutes in the DTTP technique. In addition, DTTP may not be sufficiently reliable for E. coli since their dispersion time is less than the cut-off of 120 minutes.

The Effect of Ammonium Ions on the Corrosion Resistance of Zinc - Aluminium and Zinc - Aluminium - Magnesium Coated Steel

Galvanizing is the process of applying a protective zinc or zinc alloy coating to steel or iron which acts as a sacrificial anode to protect the underlying steel or iron. These coatings are mainly used in the automotive and construction industry where corrosive environments will be encountered. There has been a drive to improve the performance of galvanised coatings through alloy additions, initially Al (5 – 55 wt%) and more recently through the development of Zn - Al - Mg coatings. Here, Al and Mg are added as minority additions (typically 1 - 3 wt %) to improve corrosion resistance allowing thinner coatings to be used thus saving material, weight and cost. The assessment of the corrosion performance of such coatings is often evaluated through standardised tests. An industry standard test known as Prohesion or ASTM G85 Annex A5 is carried out on metallic coatings as it is thought to better represent long-term natural exposure than a regular salt spray test. The Prohesion test is a cyclic accelerated corrosion test using an electrolyte of 0.4% Ammonia sulphate and 0.05% sodium chloride (NaCl).This investigation aims to determine the effects on the corrosion performance of zinc-aluminium alloys and zinc-aluminium-magnesium alloys of varying compositions immersed in Prohesion solution. Previous work on this topic suggests that magnesium containing alloys were particularly susceptible to accelerated corrosion in Prohesion solution. Here, several zinc based alloys were immersed in Prohesion solution as well as a control 0.45% sodium chloride solution and subjected to electrochemical testing. The electrochemical techniques used to test said substrates are linear polarisation resistance (LPR), scanning vibrating electrode technique (SVET) and time-lapse optical microscopy (TLM) all for 24 hours. The alloys that underwent testing are shown in Table 1. Table 1: Table showing the composition of substrates used.Sample number Composition 1 Zn-<0.2wt%Al 2 Zn-5wt%Al 3 Zn-55wt%Al 4 Zn-1.2wt%Al-Zn1.2wt%Mg 5 Zn-3.5wt%Al-3wt%Mg 6 Zn-6wt%Al-3wt%Mg The results from the TLM showed differences in the corrosion mechanism that is dependent on the alloy composition and electrolyte. Generally, all substrates immersed in the control 0.45% NaCl showed localised corrosion with distinct anodes developing surrounded by a ring of corrosion product and white rust is observed on the surface of the substrate. The initial site of anodic attack in the microstructure was dependent on the alloy composition. However, when immersed in the Prohesion solution significant differences were observed dependent on the alloy composition. For Zn-Al-Mg coatings rapid attack of zinc-magnesium phases was observed especially in the eutectic regions followed by aggressive attack of the entire exposed surface of the alloy within 6 hours. Additionally, no corrosion product rings formed. For Zn-Al alloy coatings, the zinc phases were preferentially attacked with the aluminium containing phases appearing to be resistant to corrosion during the duration of the tests. No white rust was seen on the surface of the substrates and instead a dark surface/corrosion product film was observed. The corrosion rate from the LPR data measured at hourly intervals over 24 hours increases when substrates are immersed in Prohesion solution compared to NaCl, with the most noticeable increase seen when there are zinc-magnesium phases present within the alloy. The largest increase in corrosion rate mm per year (mmpy) was seen in sample 5 with it increasing from 0.15 mmpy after 24 hours in the control solution to 0.55 mmpy in the Prohesion solution after 24 hours. The same is observed with the SVET results showing that the ammonium ions are having a significant damaging effect on the Zn-Al-Mg metallic coatings. The proposed corrosion mechanism for Zn-Al-Mg alloy coatings in Prohesion solution is preferential dissolution of MgZn2 in eutectic phases due to reactivity of Mg producing Mg(OH)2 which is a highly basic hydroxide. Due to the basic nature of Mg(OH)2, Ammonium (NH4 +) cations react with Mg(OH)2 producing Mg2+ and Ammonia (NH3).Mg(OH)2+2NH4 +=Mg2++2NH3+2H2OZn(OH)2 does not undergo this reaction to the same extent as it hydrolyses in water (producing H+) therefore is not basic enough to support much NH3 production. Neutral NH3 reacts with metallic Zn or Zn2+ compounds to produce highly soluble zinc (II) tetra-ammine complex cations. Therefore, the protective Zn and Mg compounds forming at the metal surface will dissolve and the surface will become activated. This is demonstrated by the lack of white rust shown from samples immersed in the Prohesion solution. Al phases are resistant to NH3 hence the better performance of Zn-Al coatings and the preferential attack of the Zn in the Zn-Al timelapse microscopy.

Role of Mass Transport in the Deposition, Growth, and Transformation of Calcium Carbonate on Surfaces at High Supersaturation.

We demonstrate how combined in-situ measurements and finite element method modeling can provide new insight into the relative contribution of mass transport to the growth of calcium carbonate on two model surfaces, glass and gold, under high-supersaturation conditions relevant to surface scaling. An impinging jet-radial flow system is used to create a high-supersaturated solution at the inlet of different cells: an optical microscope cell presenting a glass surface for deposition and quartz crystal microbalance (QCM) and in-situ IR spectroscopy cells, both presenting a gold surface. The approach described is quantitative due to the well-defined mass transport, and both time-lapse optical microscopy images and QCM data are analyzed to provide information on the growth kinetics of the calcite crystals. Initially, amorphous calcium carbonate (ACC), formed in solution, dominates the deposition process. At longer times, the growth of calcite is more significant and, on glass, is observed to consume ACC from the surface, leading to surface regions depleted of ACC developing around calcite microcrystals. On Au, the mass increase becomes linear with time in this region. Taken together, these microscopic and macroscopic measurements demonstrate that calcite growth has a significant component of mass transport control at high supersaturation. Finite element method (FEM) simulations of mass-transport-limited crystal growth support the strong mass transport contribution to the growth kinetics and further suggest that the observed growth must be sustained by more than just the Ca2+ and CO32– in solution, with dissolution/direct attachment of ACC and/or ion pairs also contributing to the growth process.

Breast radiotherapy with kilovoltage photons and gold nanoparticles as radiosensitizer: An in vitro study.

PurposeWe investigated the dose enhancement and internalization of gold nanoparticles (AuNPs) used as a radiosensitizer agent for rotational radiotherapy of breast cancer using a kilovoltage (kV) X‐ray beam.MethodsHuman breast cancer cells MDA‐MB‐231 were incubated with or without 100 μg/mL (4.87 nM) or 200 μg/mL (9.74 nM) 15 nm AuNPs and irradiated with 100 kV, 190 kV, or 6 MV X‐rays. To assess the toxicity of the AuNPs, we performed a Sulforhodamine B assay. Using atomic absorption spectroscopy, scanning electron microscopy, transmission electron microscopy, and time‐lapse optical microscopy (rate of 2 frames per minute), we carried out a quantitative assessment of the amount of gold internalized by MDA‐MB‐231 cells and a characterization of the static and dynamical aspects of this internalization process.ResultsNo effect of AuNPs alone was shown on cell viability. Time‐lapse optical microscopy showed for the first time AuNPs cellular uptake and the dynamics of AuNPs internalization. Electron microscopy demonstrated AuNPs localization in endosomal vesicles, preferentially in the perinuclear region. After irradiation at doses up to 2 Gy, cell survival fraction curves showed increased mortality with AuNPs, with respect to irradiation without AuNPs. The highest effect of radioenhancement by AuNPs (at 9.74 nM AuNPs concentration) was observed at 190 kV showing a dose enhancement factor of 1.33 ± 0.06 (1.34 ± 0.02 at 100 kV), while at 6 MV it was 1.14 ± 0.06.ConclusionsThe observed radio‐sensitization effect is promising for future radio‐enhanced kV radiotherapy of breast cancer and quantitatively in the order of previous observations for 15 nm AuNPs. These results of a significant dose enhancement were obtained at 15 nm AuNPs concentration as low as several nanomolar units, at dose levels typical of a single dose fraction in a radiotherapy session. Dynamical behavior of the 3D spatial distribution of 15 nm AuNPs outside the nucleus of single breast cancer cell was observed, with possible implications for future models of AuNPs sensitization.

Deciphering porosity clogging at barrier interfaces in deep geological repositories for radioactive waste

Abstract. The disposal of spent nuclear fuels and high-level radioactive wastes in deep geological repositories represents one of the greatest scientific-technical and societal challenges of our times. Most disposal concepts rely on a multibarrier system, consisting of a combination of engineered materials, geotechnical and geological barriers to provide a safe containment of the radioactive waste to protect humans and the environment against dangers arising from ionizing radiation. A reliable safety assessment of a deep geological repository over assessment time scales of several 100 000 years requires a profound and comprehensive understanding of the complex coupled physical (thermal, hydraulic, mechanical), chemical and biogeochemical (THM/CB) processes that govern the long-term evolution of the repository system. As a result of thermal and chemical gradients at the interfaces of different components and materials of the multi-barrier system (e.g. interfaces between metallic waste containers and bentonite backfill or between structural concrete and clay host rock), mineral dissolution and precipitation reactions are promoted; thus the (local) porosity, the volume filled with gas and/or water, can increase or decrease leading to changes in the macroscopic transport properties of the respective media. Although a reduction of the porosity (porosity clogging) appears to be desirable to inhibit radionuclide migration, it can also be detrimental, particularly in the case of gas pressure build-up due to canister corrosion or bacterial activity. So far, porosity clogging at barrier interfaces and associated consequences on solute or gas transport remain poorly understood; currently used mathematical descriptions of porosity clogging in reactive transport codes usually fail to capture respective experimental observations (Chagneau et al., 2015; Deng et al., 2021). In this context, we are developing a “lab-on-a-chip” set-up, which combines time lapse optical microscopy imaging and in operando Raman spectroscopy (Poonoosamy et al., 2019, 2020) to determine (i) whether complete clogging is possible and permanent, (ii) which parameters control the porosity clogging and (iii) which changes in transport properties of porous media are induced due to porosity clogging. Our approach comprises micronized counterdiffusion experiments with in situ visualization and monitoring of the evolution of mineralogy and microstructure/pore architecture with time. Complementary pore scale modelling will be used to derive key relationships that describe changes in transport properties due to mineral precipitation-induced porosity clogging. This approach will help to improve reactive transport codes and their predictive capabilities thus enhancing confidence and reduce uncertainties in long-term predictions, leading to more realistic descriptions of the evolution of complex repository systems.

(Invited) Selective Response of Magnesium Corrosion Inhibitors Incorporated in PEO Coatings

Corrosion inhibitors are the integral components of protective coatings used by aerospace, automotive, and consumer goods industries. Despite recent achievements in discovering new, effective magnesium corrosion inhibitors [1,2], the quest to replace the highly effective but carcinogenic Cr(VI) from corrosion inhibiting processes continues and has now become urgent. Plasma electrolyte oxidation (PEO), also known as Micro-Arc Oxidation (MAO), is widely used to form coatings on magnesium with reasonable wear and corrosion resistant properties. Unexpectedly, we found out that not all of the corrosion inhibitors, efficient for a bare magnesium alloy, can effectively inhibit its corrosion once loaded in PEO coating. In an effort to unravel the mechanism behind this phenomenon and to formulate the criteria for selecting inhibitors that maintain their efficiency in PEO coatings, a number of inhibitor-loaded PEO coatings have been synthesized. Several electrochemical and surface analysis techniques were used to thoroughly characterize the reference PEO, those loaded with the corrosion inhibitors, and compare all PEO-based coatings with the Chromate Conversion Coating as an industrial reference.Electrochemical Impedance Spectroscopy (EIS) was the main method to rank the corrosion protection properties of the inhibitor-loaded PEO coatings. The inhibitors were ranked based on their ability to suppress the corrosion onset of inhibitor-loaded PEO coating. ToF-SIMS and XPS characterization exposed that specific magnesium corrosion inhibitors can promote fast formation of conversion layer that assures further corrosion inhibition. This, however, does not occur on the ceramic-like surface of PEO that, at first approximation, can be represented as MgO/Mg(OH)2. On the contrary, effective adsorption or precipitation inhibitors typically maintain their inhibiting efficiency once loaded in PEO coatings.In particular, one of the salicylate derivatives, 4-methyl-salicylate, shown to be prone to chemisorption on Mg [3,4], revealed high inhibiting efficiency on both: bare and PEO treated magnesium. The SVET measurements coupled with time lapse optical microscopy revealed the initial stages of PEO degradation followed by the outbreak of filiform corrosion and its decay observed only for the sample protected by the inhibitor-loaded PEO coating. The reference PEO sample resisted the corrosion outbreak for ca. 45h, which is only 10 hours less that the inhibitor-loaded PEO sample, but once the filiform corrosion started it remained in its active phase for the duration of the measurement, 94 hours. Surprisingly, the sample protected by the Chromate Conversion Coating revealed persistent, albeit moderate, corrosion activity throughout the measurement. In contrast, the sample containing the corrosion inhibitor, 4-methyl-salicylate, demonstrated the active corrosion protection evident from the decay of the initial filiform corrosion. The second event of filiform corrosion also faded away after some hours of activity. This clearly demonstrates the active corrosion protection provided by the inhibitor-loaded PEO coating. The inhibitor loaded PEO coating revealed a corrosion protection superior to that provided by the industrially accepted Chromate Conversion Coating.The criteria of an effective corrosion inhibitor for PEO-coated magnesium will be discussed based on the inhibitor interaction with the bare magnesium and PEO coating. Acknowledgement The authors acknowledge the financial support of the following projects: H2020 Clean Sky 2 “Almagic” Grant Agreement Number 755515, and HZG IDEA project MMDi. Di Mei thanks China Scholarship Council for the award of fellowship and funding (No. 20160704051).

Effect of degradation products of iron-bioresorbable implants on the physiological behavior of macrophages in vitro

The degradation of bioresorbable metals in vivo changes the physicochemical properties in the environment of an implant, such as a stent in the artery wall, and may induce the alteration of the functions of the surrounding cells. The Fe-degradation, from bioresorbable stents, is a particularly intricate process because it leads to the release of soluble (SDP) and insoluble degradation products (IDP) of varied composition. Macrophages are involved in the resorption of the exogenous agents coming from degradation of these materials. In the present work an Fe0 ring, made with a pure Fe wire, in contact with macrophage cell cultures was used to simulate the behaviour of a biodegradable Fe-based implant in a biological environment. Non-invasive time-lapse optical microscopy was applied to obtain images of macrophages exposed to Fe-degradation products, without using staining to avoid distortions and artefacts. It was noticed that as metal degraded, the IDP formed in situ accumulated close to the Fe0 ring. In this zone, the macrophages showed a dynamic process of uptake of dark Fe-containing products, confirmed by SEM-EDX. These macrophages showed alterations in the morphology and decrease in the motility and viability. The inability of the macrophages to move and to degrade the engulfed products caused a long persistence of IDP in the zone closest to the metal. The deleterious effects of IDP accumulated close to the ring, were significantly worse than those observed in the experiments made with (1) concentrated salt solutions (Fe3+ salt 3 mM), with the same amount of precipitates but uniformly distributed in the well, and (2) diluted salt solutions (Fe3+ salt 1 mM) with mainly soluble species. The results were confirmed by standard staining protocols that revealed dead cells close to the Fe0 ring and oxidative stress in cells exposed to both soluble and insoluble species.

A biphasic growth model for cell pole elongation in mycobacteria

Mycobacteria grow by inserting new cell wall material in discrete zones at the cell poles. This pattern implies that polar growth zones must be assembled de novo at each division, but the mechanisms that control the initiation of new pole growth are unknown. Here, we combine time-lapse optical and atomic force microscopy to measure single-cell pole growth in mycobacteria with nanometer-scale precision. We show that single-cell growth is biphasic due to a lag phase of variable duration before the new pole transitions from slow to fast growth. This transition and cell division are independent events. The difference between the lag and interdivision times determines the degree of single-cell growth asymmetry, which is high in fast-growing species and low in slow-growing species. We propose a biphasic growth model that is distinct from previous unipolar and bipolar models and resembles “new end take off” (NETO) dynamics of polar growth in fission yeast.

Effect of Nanoparticles on the Bulk Shear Viscosity of a Lung Surfactant Fluid.

Inhaled nanoparticles (<100 nm) reaching the deep lung region first interact with the pulmonary surfactant, a thin lipid film lining the alveolar epithelium. To date, most biophysical studies have focused on particle-induced modifications of the film interfacial properties. In comparison, there is less work on the surfactant bulk properties and on their changes upon particle exposure. Here we study the viscoelastic properties of a biomimetic pulmonary surfactant in the presence of various engineered nanoparticles. The microrheology technique used is based on the remote actuation of micron-sized wires via the application of a rotating magnetic field and on time-lapse optical microscopy. It is found that particles strongly interacting with lipid vesicles, such as cationic silica (SiO2, 42 nm) and alumina (Al2O3, 40 nm) induce profound modifications of the surfactant flow properties, even at low concentrations. In particular, we find that silica causes fluidification, while alumina induces a liquid-to-soft solid transition. Both phenomena are described quantitatively and accounted for in the context of colloidal physics models. It is finally suggested that the structure and viscosity changes could impair the fluid reorganization and recirculation occurring during breathing.

A novel apparatus for time-lapse optical microscopy of gelatinisation and digestion of starch inside plant cells

A new instrument was developed for time-lapse optical microscopy of a cohort of individual food particles, which we denote as ParCS (Particle Cohort Study). The cohort can be observed through each stage of simulated hydrothermal processing, followed by in vitro gastrointestinal digestion. Instrument capabilities include the quantification of cooking and digestion dynamics of starch trapped inside isolated cotyledon cells from navy beans. The cells, contained within a flow-through chamber, were cooked at 90°C and subsequently subjected to simulated gastric digestion and simulated small-intestinal digestion. Little or no cell expansion was observed, and the cells remained intact, i.e., the cell walls showed no signs of rupture throughout the entire process. In contrast, intracellular starch granules partially swelled and gelatinised with a maximum relative granule area of approximately 2.4 at the end of cooking. During the small intestinal digestion, cellular contents were observed to visually “shrink” radially inwards towards the centre of the cells, implying that starch hydrolysis by pancreatic α-amylase had occurred inside the cells. Kinetic modelling of this shrinking process showed that the cells underwent amylolysis of cellular contents at different rates. This latter finding demonstrates that our new technique allows quantitative characterisation of starch gelatinisation and digestion inside cotyledon cells at the single-particle scale and may be used to test mechanistic hypotheses.

Response to Blast-like Shear Stresses Associated with Mild Blast-Induced Brain Injury

Toward the goal of understanding the pathophysiology of mild blast-induced traumatic brain injury and identifying the physical forces associated with the primary injury phase, we developed a system that couples a pneumatic blast to a microfluidic channel to precisely and reproducibly deliver shear transients to dissociated human central nervous system (CNS) cells, on a timescale comparable to an explosive blast but with minimal pressure transients. Using fluorescent beads, we have characterized the shear transients experienced by the cells and demonstrate that the system is capable of accurately and reproducibly delivering uniform shear transients with minimal pressure across the cell culture volume. This system is compatible with high-resolution, time-lapse optical microscopy. Using this system, we demonstrate that blast-like shear transients produced with minimal pressure transients and submillisecond rise times activate calcium responses in dissociated human CNS cultures. Cells respond with increased cytosolic free calcium to a threshold shear stress between 8 and 21 Pa; the propagation of this calcium response is a result of purinergic signaling. We propose that this system models, in vitro, the fundamental injury wave produced by shear forces consequent to blast shock waves passing through density inhomogeneity in human CNS cells.

Time-lapse confocal imaging datasets to assess structural and dynamic properties of subcellular nanostructures

Time-lapse optical microscopy datasets from living cells can potentially afford an enormous amount of quantitative information on the relevant structural and dynamic properties of sub-cellular organelles/structures, provided that both the spatial and temporal dimensions are properly sampled during the experiment. Here we provide exemplary live-cell, time-lapse confocal imaging datasets corresponding to three sub-cellular structures of the endo-lysosomal pathway, i.e. early endosomes, late endosomes and lysosomes, along with detailed guidelines to produce analogous experiments. Validation of the datasets is conducted by means of established analytical tools to extract the structural and dynamic properties at the sub-cellular scale, such as Single Particle Tracking (SPT) and imaging derived Mean Square Displacement (iMSD) analyses. In our aim, the present work would help other researchers in the field to reuse the provided datasets for their own scopes, and to combine their creative approaches/analyses to similar acquisitions.

Water-Mediated Structural Transformations of CuII 5-Halonicotinates Coordination Networks with Distinct Mechanisms.

Currently, no unequivocal evidence is given for elucidation of "black box" during the structural transformations of dynamic crystalline materials. Here, three types of mechanisms are revealed for such transformations through X-ray diffraction and optical microscopy; namely, single-crystal to single-crystal (SC-SC), as well as "core-to-core" and "core-on-shell" processes. As confirmed by time-lapse optical microscopy, the latter two cases can be properly ascribed as partial recrystallization processes, while the former one is a continuous process with two different crystal lattices simultaneously maintained in one single crystal. Interestingly, these three distinct pathways can be exquisitely realized by changing only the halogen substituent (from -F, -Cl, to -Br) of the organic ligands in the coordination supramolecular systems.

Investigation into the Corrosion Mechanisms and Effective Inhibitor Additions for Zinc-Magnesium-Aluminium (ZMA) Alloys

The use of zinc-magnesium-aluminium (ZMA) alloys is increasing in popularity within the coated steel industry due to the potential advantages it can offer. As such, a collaboration of work involving three differing ZMA alloy coated steels were studied using the scanning vibrating electrode technique (SVET) and novel in situ time-lapse optical microscopy whilst immersed in a 1% NaCl electrolyte, facilitating the investigation of each microstructure. The collation of data demonstrated preferential corrosive attack of the eutectic phases amongst each of the alloy compositions, and more specifically the MgZn2 lamellae, succeeded by the corrosion of Zn-rich dendrites. The use of SVET enabled an estimation of mass loss from each alloy, and when correlated with the time-lapse data, revealed a reduction in magnitude of corrosion with increasing content of Mg and Al alloy additions. The improvement in corrosion performance was likely a result of increased levels of both MgO and Al2O3 existing at the alloy surface, thereby restraining the corrosion event kinetics. A corrosion inhibitor in the form of Na3PO4 introduced to 1% NaCl electrolyte at varying concentrations exhibited marked effects on the corrosion of the selected ZMA alloy, as evidenced by both SVET and time-lapse microscopy data sets. Approximations based on the measured SVET data showed that sodium phosphate additions diminished the corrosion activity, the extent of which was dependant on the chosen concentration. The time-lapse images establish suitable correlation with the estimated mass loss results at pH 7, whereby images indicated phosphate precipitation at anodic sites, and thus their passivation. Based on these observations, it can be realised that sodium phosphate inhibitor additions have an appreciable effect on corrosion behaviour within this particular alloy system.

Axon zippering in neuronal cell culture and its biophysical modeling

During neural development, the growing axons frequently adhere to each other and form tight bundles. This process contributes to the setting up of correct connectivity in, e.g., the mammalian olfactory system [1]. Here we report an experimental and theoretical investigation of axon bundling in olfactory epithelium explants; in this system, the dynamics of axon-axon interactions can be directly examined. Our analysis is based on time-lapse optical microscopy observations of axon growth in primary cell culture. Olfactory epithelium explants from mouse embryos (day 13-14) were cultured on laminin substrate for two days and then recorded using DIC videomicroscopy for up to 24 hours. The growing axons established a dense network within which large bundles of axons were progressively formed. This dynamics was driven by numerous fast processes, during which the length of the segment along which two axons adhere to each other was increased. Similar processes were inferred in 1982 from electron-microscopy-based analysis of sensory neurites in Xenopus embryos [2], but have been very rarely studied in the subsequent literature. We digitized a collection of 30 spontaneous zippering processes observed in the developing axon network. During zippering, the vertex (i.e. the point where the two axons meet) advances with a typical velocity of 0.2 to 1 µm/min. The distance between immediate position of the vertex and the final equilibrium position decreases either linearly with sudden arrest or exponentially in time. We observed similar behaviour, at velocities of 1 to 3 µm/min, for zippering events induced during micro-manipulation experiments. To explain these observations, we developed a simplified biophysical model that includes the adhesive force between the axons and the mechanical tension in the axons, as well as adhesion and anisotropic friction with the substrate. A static zipper is obtained if the tensile and adhesive forces are balanced. The model predicts that the linear regime of convergence to equilibrium can arise from a perpendicular outer force acting on one of the axons, while the exponential regime is a result of sudden change in axial tension of the axon. The parameters of the model were estimated from calibrated micro-manipulations of the culture, using a biotinylated red blood cell as force transducer [3]. These experiments allowed us to measure the tension within the axon shaft (typically about 1 nN) and deduce the force of adhesion between two axon shafts (of order 100 pN). To account for additional factors, we implemented a molecular-dynamics-type simulation. The beads-and-springs representation of each axon permits to include bending rigidity and allows for an inhomogeneous distribution of tension along the axon. To implement the interaction between axons, we adapted results from contact mechanics [4] for cylindrical bodies. This permits to avoid artifacts that would result from direct adhesive interaction between beads. We compare the dynamics of zippering in this detailed model to the predictions of the simplified analytical model.

Circular random motion in diatom gliding under isotropic conditions

How cells migrate has been investigated primarily for the case of trajectories composed by joined straight segments. In contrast, little is known when cellular motion follows intrinsically curved paths. Here, we use time-lapse optical microscopy and automated trajectory tracking to investigate how individual cells of the diatom Nitzschia communis glide across surfaces under isotropic environmental conditions. We find a distinct kind of random motion, where trajectories are formed by circular arcs traveled at constant speed, alternated with random stoppages, direction reversals and changes in the orientation of the arcs. Analysis of experimental and computer-simulated trajectories show that the circular random motion of diatom gliding is not optimized for long-distance travel but rather for recurrent coverage of limited surface area. These results suggest that one main biological role for this type of diatom motility is to efficiently build the foundation of algal biofilms.

Biophysical and morphological effects of nanodiamond/nanoplatinum solution (DPV576) on metastatic murine breast cancer cells in vitro

Nanoparticles have recently gained increased attention as drug delivery systems for the treatment of cancer due to their minute size and unique chemical properties. However, very few studies have tested the biophysical changes associated with nanoparticles on metastatic cancer cells at the cellular and sub-cellular scales. Here, we investigated the mechanical and morphological properties of cancer cells by measuring the changes in cell Young’s Modulus using AFM, filopodial retraction (FR) by time lapse optical light microscopy imaging and filopodial disorganization by high resolution AFM imaging of cells upon treatment with nanoparticles. In the current study, nanomechanical changes in live murine metastatic breast cancer cells (4T1) post exposure to a nanodiamond/nanoplatinum mixture dispersed in aqueous solution (DPV576), were monitored. Results showed a decrease in Young’s modulus at two hours post treatment with DPV576 in a dose dependent manner. Partial FR at 20 min and complete FR at 40 min were observed. Moreover, analysis of the retraction distance (in microns) measured over time (minutes), showed that a DPV576 concentration of 15%v/v yielded the highest FR rate. In addition, DPV576 treated cells showed early signs of filopodial disorganization and disintegration. This study demonstrates the changes in cell stiffness and tracks early structural alterations of metastatic breast cancer cells post treatment with DPV576, which may have important implications in the role of nanodiamond/nanoplatinum based cancer cell therapy and sensitization to chemotherapy drugs.

Nanoscale Imaging and Mechanical Analysis of Fc Receptor-Mediated Macrophage Phagocytosis against Cancer Cells

Fc receptor-mediated macrophage phagocytosis against cancer cells is an important mechanism in the immune therapy of cancers. Traditional research about macrophage phagocytosis was based on optical microscopy, which cannot reveal detailed information because of the 200-nm-resolution limit. Quantitatively investigating the macrophage phagocytosis at micro- and nanoscale levels is still scarce. The advent of atomic force microscopy (AFM) offers an excellent analytical instrument for quantitatively investigating the biological processes at single-cell and single-molecule levels under native conditions. In this work, we combined AFM and fluorescence microscopy to visualize and quantify the detailed changes in cell morphology and mechanical properties during the process of Fc receptor-mediated macrophage phagocytosis against cancer cells. Lymphoma cells were discernible by fluorescence staining. Then, the dynamic process of phagocytosis was observed by time-lapse optical microscopy. Next, AFM was applied to investigate the detailed cellular behaviors during macrophage phagocytosis under the guidance of fluorescence recognition. AFM imaging revealed the distinct features in cellular ultramicrostructures for the different steps of macrophage phagocytosis. AFM cell mechanical property measurements indicated that the binding of cancer cells to macrophages could make macrophages become stiffer. The experimental results provide novel insights in understanding the Fc-receptor-mediated macrophage phagocytosis.

In situ monitoring of the microstructural corrosion mechanisms of zinc–magnesium–aluminium alloys using time lapse microscopy

A novel technique has been developed that enables in situ monitoring of the microstructural wet corrosion mechanisms of zinc–(1–2 wt.% magnesium)–(1–2 wt.% aluminium) galvanising alloys using time lapse optical microscopy. The technique enabled the imaging of the progression of anodic attack, the development of corrosion product rings radially to the anode and pH gradients between anodes and cathodes using an indicator. It was found that corrosion initiated in the binary and ternary eutectic regions within the microstructure of the alloy with preferential de-alloying of MgZn 2 lamellae. After eutectic dissolution, anodic attack proceeded on the primary zinc rich dendrites.