Lymphocytes alternate between phases of individual migration across tissues and phases of clustering during activation and function. The range of lymphocyte motility behaviors and the identity of the factors that govern them remain elusive. To explore this point, we here collected unprecedented statistics pertaining to cell displacements, cell:matrix and cell:cell interactions using a model B cell line as well as primary human B lymphocytes. At low cell density, individual B lymphocytes displayed a high heterogeneity in their speed and diffusivity. Beyond this intrinsic variability, B lymphocytes adapted their motility to the composition of extra-cellular matrix, adopting slow persistent walks over collagen IV and quick Brownian walks over fibronectin. At high cell density, collagen IV favored the self-assembly of B lymphocytes into clusters endowed with collective coordination, while fibronectin stimulated individual motility. We show that this behavioral plasticity is controlled by acto-myosin dependent adhesive and Arp2/3-dependent protrusive actin pools, respectively. Our study reveals the adaptive nature of B lymphocyte motility and group dynamics, which are shaped by an interplay between and cell:matrix and cell:cell interactions. Within tissues, lymphocytes can organize into swarms and clusters to sustain specific steps of activation and function5. Group organization has also been reported to condition tissue residency of memory lymphocyte populations7. Furthermore, our recent in vitro observations have revealed that the assembly of B and T lymphocytes into clusters confers collective properties leading to distinct chemotactic prowess8. Understanding the parameters that govern the transition from individual to group behaviors in lymphocytes would provide insight into the mechanisms that determine collective cell behaviors common to many cellular systems. A key question is to disentangle the relative contribution of cell intrinsic versus extrinsic factors in the emergence of collective cell behaviors. To tackle this question, we here reasoned that the characterization of individual cell motility properties and encounter dynamics would explain the emergence of collective organization.Lymphocytes represent a unique model to study cell motility because they may adopt a vast array of behaviors. These immune cells patrol the organism as isolated individuals endowed with high motility properties favoring the search for pathogens or pathogen-derived determinants9. Lymphocyte migrate as amoeboid cells with patterns characterized as a Brownian random walk10, a persistent random walk12 or a Lévy walk13. While both walking behaviors might accommodate local search and tissue diffusion, it is not yet elucidated which parameters might govern their onset14. Environmental factors such as extra-cellular matrix (ECM) components affect the motility behavior of lymphocytes15. Indeed, lymphocytes can adopt walking versus sliding crawling modes depending on substrate attachment17. A recent study revealed the presence of “tissue hotspots” that locally influence cell motility properties to favor local search18. Furthermore, spatial and temporal changes in ECM composition have been demonstrated to control processes such as lymphocyte recruitment, survival and differentiation20. Altered expression of fibronectin and collagens I and IV in multiple myeloma is associated to progressive accumulation of malignant lymphocytes in the bone marrow at the different stages of disease progression21.As individual cells, lymphocytes face the challenge of patrolling large tissue areas, while scanning locally to discriminate between cells presenting different antigenic determinantsin vitro experimental framework and a dedicated tracking algorithm, we produce unprecedented statistics on B lymphocyte motility properties that highlight both individual heterogeneity and flocking dynamics. Our study reveals that B lymphocyte populations are composed of individuals with distinct diffusion properties that are modulated by interactions with the extracellular matrix. Fibronectin favors B lymphocyte adhesion and increases the proportion of individual cells with high-speed characteristics and chemotactic ability, thereby favoring isolated patrolling. In contrast, reduced adhesion over collagen IV favors group assembly and flocking activity that translate into collective chemotaxis. This study also highlights that actin cytoskeleton remodeling is a key integrator of B lymphocyte adaptive motility properties. Indeed, we identify distinct actin modules that coordinate adhesion to the matrix and protrusive activity involved in cell displacement and encounters with neighbors. Together, our study identifies external cues and intrinsic pathways that explain the wide range of B lymphocyte motility behaviors and control the shift between single versus collective B lymphocyte dynamics.The aim of the present study is to characterize the range of B lymphocyte motility patterns at the single cell level and to decipher which interactions and behavioral mechanisms are involved in the emergence of coordinated groups. Using a well-controlled 22 or fibronectin, a stromal matrix fibrillar component23. These ECM components were chosen for their in vivo relevance and distinct effects in pathological processes such as wound healing in the spinal cord20 and multiple myeloma progression in the bone marrow21. The JY cells were checked for the expression of the alpha chains of the integrins known to recognize collagen IV  and fibronectin (CD49d). Notably, JY cells expressed homogeneous integrin levels, which were comparable to those of primary B cells from the peripheral blood of 3 donors  and over prolonged time periods (up to 12 h) using bright-field illumination to avoid phototoxicity. A dedicated algorithm based on local light contrast changes around the cell perimeter was designed to automatically recognize and track hundreds of cells on different video-recordings yielding robust motility statistics (see Methods). B lymphocytes display distinct motility behaviors over the two matrices (Supplementary Video −1 range for most of the cells), while it alternates between slow and high values for most of the cells over fibronectin (0 to 20 μm min−1 range)  Fig. . Such vants Fig.  shows a nts Fig.  shows th17. Cells that display large and sustained matrix attachment fail to displace efficiently, probably because of a reduced ability to detach from the matrix. Together, these observations indicate that the heterogeneity of B lymphocytes diffusive properties is mirrored by distinct actin-rich patterns of matrix attachment.To further investigate which motility parameters might explain the distinct diffusive characteristics of B lymphocytes over the 2 model matrices, we measured, for each cell, the mean instantaneous speed (time resolution of 5 sec) and the mean displacement over 5-min periods. Our analysis reveals that the distribution of the mean instantaneous speeds of individual B lymphocytes is very similar over the 2 matrices Fig. , while tTo further characterize the diffusion properties of B lymphocytes over the two model matrices, we analyzed the distribution of individual displacement lengths normalized by their mean value, for time lags of 5, 10, 20, 40 minutes Fig. . In both−2) over collagen IV or fibronectin and followed for 12 h with our tracking algorithm, for which a cluster size color-coded classifier and a fusion/fission counter were implemented  and Arp2/3-dependent actin branching (CK869). Over collagen IV, both the blockade of ROCK1 and Arp2/3 reduce cell diffusion  favors aggregation of cells into radial clusters via their uropods in a way reminiscent of previous observations27. On the other hand, B lymphocytes over fibronectin fail to engage in stable cell-cell contacts. Our data point to a contact inhibition of locomotion mechanism, as described in other cells44. Our data show that the nature of the matrix, rather than its local concentration conditions specific cell behaviors. This calls for further exploration of whether the physical properties of the matrices, rather than their biochemical nature or the specificities of the corresponding adhesion receptors might explain the distinct cell behaviors. Interestingly, cell-matrix and cell-cell interactions are associated to seemingly distinct actin cytoskeleton modules. We therefore speculate that the interplay between cell-matrix and cell-cell interactions is controlled via a crosstalk between those 2 actin-rich areas. Along this concept, the different adhesive areas over the 2 matrices are associated to distinct protrusive activities impacting on the establishment of cell-cell contacts. This is reinforced by the finding that Arp2/3 is involved in cell-cell adhesion stabilization, rather than in matrix interaction. Given its role in protrusion stability and LFA-1 activation46, Arp2/3 could prime lamellipodia-like extensions that favor extensive contacts between cells, further eliciting LFA-1 driven homotypic adhesions as described for leukemic cells7. This is in agreement with the recent finding that the lamellipodium of lymphocytes is dispensable for cell traction during motility, but holds an exploratory role47.Our study further highlights that at high cell densities, B lymphocytes alternate between individual and collective behaviors. Importantly, cell-matrix interaction appears to affect cell-cell interaction dynamics, which control the assembly into clusters. Such interplay between cell-matrix and cell-cell interactions has previously been described for neural crest cells49. Our study indicates that B lymphocyte clusters form cellular hubs that could combine the necessity for B lymphocytes to cooperate during activation and/or function, with the necessity to scan actively tissue surfaces and to recirculate. Indeed the equilibrium between clusters and individual cells is highly dynamical since groups are undergoing active fusion and fission events allowing most cells to temporally contribute to clusters. Like for the grazing dynamics in flocks of sheep50, B lymphocyte populations appear to balance the conflicting needs of spreading and gathering. Whereas Merino sheep alternate between group spreading and aggregation phases, B lymphocytes establish a rather stable equilibrium between compact groups and freely moving cells at the population level. Those properties might contribute to the ability of B lymphocytes to both circulate as isolated cells and to assemble into dense follicles in lymphoid organs. Additionally, the specific nature of the reticular ECM fibers composing the scaffold of the B cell follicles is expected to play a key role in the compartmentalization and dynamic behaviors of those cells40. A further challenge will be to investigate how the multiple 3D cell-matrix adhesions and inter-cellular interactions are regulating the multi-cellular dynamics of B cell follicles.The concept that hematopoietic cells are solitary cells and migrate independently from each other has recently been challenged by reports showing that both myeloid and lymphoid cells can switch from single cell to group behaviors7. Such coordination might apply to the non-tumoral B lymphocytes studied here. In light of our studies, a possible explanation for B lymphocyte coordination over collagen IV would be that cell clusters are pulled by exchanging leader cells that transiently engage in strong attachment to the matrix. Concomitantly, pulling of the cell clusters by such leader cells would be favored by the stability of cell-cell interactions. On the other hand, under conditions favoring strong attachment of most cells, the loose cell-cell contacts are generating noise that counteracts diffusion and chemotaxis. Such noise might be explained by cell-cell frictions, contact-inhibition of locomotion, or cell-cell interactions weakening cell-matrix interaction. The importance of cell-cell interactions during collective chemotaxis over collagen IV is highlighted by our experiments with the ROCK1 inhibitor. The fact that clusters display increased chemotactic speed upon ROCK1 inhibition suggests that myosin contraction in cell clusters limits cluster compactness and onset of collective chemotaxis. This hypothesis is in line with results obtained in carcinoma cells, in which the DDR1-driven down-modulation of myosin contraction in the core of clusters is required for collective migration51. It is currently unclear whether collective chemotaxis of lymphoid cells might occur in vivo because of space constraints. We favor the view that lymphocytes might exploit collective properties to assemble in areas of high chemokine concentration, such as in the context of B cell follicles.Our study reveals the role of cell matrix interaction on the onset of B lymphocyte collective motility. Over fibronectin, B cell clusters display reduced basal motility and chemotaxis as a function of size, pointing to the lack of coordination among the cells composing the clusters. In contrast, over collagen IV, B cell clusters display increased diffusion and chemotaxis as compared to individual cells. Both behaviors are qualitatively consistent with the Einstein relation for diffusing particles submitted to an external field. Moreover, the onset of collective chemotaxis is not the consequence of the preferential aggregation of the most motile B lymphocytes. This instead points to a cell-cell coordination mechanism. Importantly, we show that the role of extracellular matrix on the onset of collective chemotaxis applies to primary B cells isolated from human peripheral blood. In the context of tumoral lymphocytes displaying a similar collective chemotaxis, we previously established that cell coordination was ensured by the turn-over of leader cells expressing high levels of chemokine receptor and protruding toward the chemokine sourceIn conclusion, our study reveals how B lymphocytes integrate cell-matrix adhesion and intercellular interactions to generate a vast array of motility behaviors at the population level. In particular, our work unveils the following rules pertaining to B lymphocyte motility: i) B lymphocytes are uniformly endowed with a local scanning activity, independently from matrix interaction; ii) space exploration by B lymphocyte populations relies on a wide distribution of individual cell diffusive abilities that is additionally tuned by matrix interaction; iii) transition from individual to schooling behaviors is highly dependent on matrix interaction. These rules provide a novel framework to explore how the complexity of physiological tissue environments or the activation status of lymphocytes are tuning their motility patterns.5 JY cells were transduced with a lentiviral vector encoding LifeAct-TagGFP2 or LifeAct-TagRFP (Ibidi) over 6 h in RPMI-1640 containing 5% FCS and polybrene (4 μg ml−1). Transfected cells were selected by culturing them in the presence of 2 μg ml−1 puromycin for 7 days, added 24 h after transfection. Bright GFP-positive cells were further selected by flow cytometry sorting. Where indicated, cells were treated with Y27632 (Abcam) at 10 μM, CK 869 (Sigma-Aldrich) at 50 μM or the equivalent concentration of DMSO (10−3). No treatment toxicity was detected in a window of 16 h. IbitreatTM (Ibidi) plastic plates were coated with 1,5 μg ml−1 collagen type IV from human placenta or 10 μg ml−1 fibronectin from human plasma (both from Sigma-Aldrich) diluted on PBS. Matrices were incubated ON at 4 °C and 1 h at 37 °C before washing with medium and cell seeding.EBV-transformed B cells (JY line) were cultured in RPMI 1640 GlutaMax, supplemented with 10% Fetal Calf Serum, minimum essential amino acids, HEPES, sodium pyruvate and Streptomycin/penicillin solution (Invitrogen) Cells were routinely screened for mycoplasm contamination using the MycoAlert mycoplasma detection kit (Lonza). For the stable expression of GFP, LifeAct-GFP, LifeAct-RFP, 1 × 106 cells ml−1 in RPMI 1640 GlutaMax, supplemented with 10% Fetal Calf Serum and stimulated for 48 h with the 5 μM CpG ODN 2006 (InvivoGen) for 48 h before performing the experiments.Buffy coats from healthy donors were obtained through the Etablissement Français du Sang  and processed following standard ethical procedures (Helsinki protocol), after obtaining written informed consent from each donor and approval by the local ethics committee (Comité de Protection des Personnes Sud-Ouest et Outremer II). B cells were isolated from buffy coats via separation by Ficoll centrifugation and a negative selection kit . Purified B cells were cultured at 5 × 10JY cells or PBMCs were washed and blocked for 20 min with PBS, 20% human serum at 4 °C. Then, cells were stained with antibodies against CD19, CD49a, CD49b or CD49d (Biolegend) at 10 µg/ml for 60 min at 4 °C. Stained cells were washed several times and analyzed using a Miltenyi MacsQuant VYB flow cytometer. Using the FlowJo software, B cells were selected by gating on the CD19 positive cells and analyzed for their expression of the CD49a, CD49b and CD49d integrin alpha-chains.3 cells in 120 µl) onto a collagen IV or fibronectin coated channel from a µ-Slide VI 0,4 (Ibidi) to reach a cell density of 50 cells mm−2. After 2 h incubation at 37 °C to allow cell attachment, cells were recorded for 12 h at a rate of 1 image each 5 s on an Eclipse TE2000-E fully-motorized inverted microscope (Nikon) and a 10x/0.45 NA objective. The microscope was equipped with an incubation chamber (OKOlab) for temperature and CO2 control. Images were acquired using a Evolve 512 emCCD camera (Photometrics) and the Metamorph software (Molecular Devices). Image processing was performed with a program written in C+ +, using the open source OpenCV library. To identify the interactions, the first step consisted in detecting and outlining the individual cells and groups. The images were binarized using Otsu method and a morphological closure was applied to reduce noise. Edge detection was implemented with the Suzuki method and the further blob labeling provided a map at any time point (t). Secondly, fusion and scission dynamics were analyzed over time by matching blobs at time t-1 with the ones found at time t. Briefly, distance, size and cell density index combination provided a score for the best blob matching. In case of several blobs with the same match, a fusion or scission event was considered. In case of a missing match, the cell (or cluster) passed to “standby mode” being considered as definitively lost if it did not reappear for 10 frames (50 sec). This method helped us to overcome the problem of over-labeling of “intermittent” cells/clusters due to faint contrasted contours. The proportion of lost cells or clusters in the analyzed video-recordings ranged between 0.44% and 1.33%, depending on ECM coating and cell density. The proportion of gaps in the analyzed tracks ranged between 1.94% and 3.10%, depending on ECM coating and cell density.JY cells were washed, resuspended in fresh medium and seeded (5 × 104 cells per well in an angiogenesis µ-plate (Ibidi) containing either uncoated wells or wells precoated with collagen IV  or fibronectin . The plate was placed under a Zeiss apotome microscope equipped with a chamber  and imaged in a bright field mode at 0, 1, 2, 4 and 6 h, using a 10x/0.45 NA objective and an Axiocam HRm Rev.3 camera. The image treatment from previous experiments was applied to recognize the perimeter of all cell groups present at each time point. Then, the mean size of individual cells was calculated for each experimental condition and each group area was referred to that value in order to estimate the number of cells composing it. We further checked that the area of clusters remained proportional to the number of cells composing them by staining and counting individual cell nuclei.JY cells were seeded at 1.2 × 103 cells mm−2. Cells were imaged for 12 h with the same settings as in the above section, except that single frames at a few time intervals were replaced by continuous imaging at a rate of 1 frame each 30 s. Where indicated, cells under were treated for 2 h before seeding with either Y27 or CK869. These drugs were kept in the cell medium during the entire recording. The same image analysis tools as above were applied to detect, count and estimate the size of cells and cell groups.JY cells were seeded over the different matrices at 10Data corresponding to the area and position of cells and cell groups recognized along the video-recordings were computed. On that basis, fusion events were identified as the disappearance of two groups in a frame linked to the appearance of a group of a larger area at the same location in the successive frame. Fission events were defined as the disappearance of a group in a frame linked to the appearance of two smaller groups at the same location in the successive frame. Both fusion and fission events were classified by size category (n) and the crossed probability of fusion and fission for the different size established as P fusion  = [Number of fusions n1 + n2 per min */] and P fission  = (Number of fissions n→n1 + n2 per min/Mean number of clusters during the 6 h recording).52 provided a tool to follow motility and clustering simultaneously. Cell speed and relative time in each size category were extracted. Speed values obtained from individual tracks shorter than 5 min were discarded. Correlation between speed and time of cluster residence/isolation represented for each cell by using GraphPad Prism.JY cells were prepared at a 1/40 GFP positive cell proportion, seeded over collagen IV or fibronectin as before and incubated for 6 h at 37 °C. The bright field and GFP channels were imaged for 6 h each 30 s on an Apotome microscope (Zeiss) and a 10x/0.45 NA objective, as described in the above section. The Image J software was used for video quantification. Briefly, the bright field channel was used to generate a binary image. Then specific masks for individual cells, groups from 2 to 5 cells and groups composed of 6 cells or more were applied and a specific signal intensity was associated to each mask. The three masks were added to recompose the original binary image, this time with an intensity code to distinguish between the three size classes. The GFP channel was used to generate a mask that identified each cell for the full video. Combination of the recomposed bright field and GFP masks and analysis with the TrackMate Image J plugin2 controlled chamber and imaged with an oil immersion 100x/1.49 NA objective. 491 nm laser was used to obtain TIRF and widefield at the cell-matrix contact focus, while IRM image was obtained by using 561 nm laser. Added to that, widefield 491 nm signal and brightfield were obtained at cell midbody focus, where cell protrusion where extended. Fixed images or live videos with a 10 s temporal resolution were taken. Relative cell attachment area was calculated by dividing TIRF LifeAct-GFP area by widefield area for each cell. Cell shape volatility was calculated using widefield LifeAct signal as reference for cell shape. Aspect ratio was calculated by dividing the major axis by the minor axis of each cell at each frame using Image J. Volatility was calculated as the standard deviation of the aspect ratio changes over consecutive frames.JY cells expressing LifeAct-GFP were prepared and seeded on collagen IV or fibronectin at low or high cell density as in previous experiments. After 2 h at 37 °C for cell adhesion and, in case, treatment action, cells were placed on a Nikon inverted spinning disk confocal microscope equipped with a back-thinned charge-coupled device camera , equipped with a temperature and CO5 cells were seeded at 103 cells mm−2 and imaged for 16 h at a frame rate of 1 image each 30 s on a Zeiss apotome microscope equipped with a chamber  and imaged in a bright field mode with a 10x/0.45 NA objective. For motility quantification, images were treated using Image J to select individual cells by their size and contrast to background and tracks over 20 min appearing after 3 h of recording were automatically registered and quantified using Trackmate. For population dynamics, individual cells, small (2 to 5 cells) and large clusters (6 or more cells) were detected and their covered surface quantified with Image J. The relative proportion of each size category was quantified and plotted with Graphpad Prism.Primary B cells were seeded over collagen IV or fibronectin and cultured for 2 h. Cells were imaged on a Nikon inverted spinning disk confocal microscope with a 100X objective for brightfield and IR illumination. Fixed images from random fields with at least 3 cells were taken and cells showing a contact area with the matrix  were quantified. For motility studies, 105 in 6 µl of culture medium) were loaded into the central transversal channel of Ibitreat 3D chemotaxis µ-slides (Ibidi) coated with collagen IV or fibronectin and incubated at 37 °C for 30 min to allow cell attachment. CCL19 0–500 ng ml−1 gradient was created following the manufacturer’s instructions. To verify the linearity of the obtained gradients, control slides were prepared using a 10% dextran-FITC solution. Analysis of fluorescence intensity profiles showed that linear gradients were established within 3 h and were stable over at least 24 h. Cell migration was recorded after 5 h by taking 1 image per 30 s for 14 h by an Apotome microscope (Zeiss) and a 5x/0.15 NA objective in temperature and CO2 controlled conditions. Obtained images were treated and binarized using Image J to create masks corresponding to individual cells, small (2 to 5 cells) and large (6 or more cells) clusters. For primary B cells, the relative paucity of clusters led us to consider only one cluster category. Migration tracks of each category were obtained using the TrackMate plugin of the FIJI software. Only tracks lasting more than 20 min were considered. Chemotaxis plots and migration parameters  were obtained with the Chemotaxis and Migration tool from Ibidi. Chemotactic speed was calculated by multiplying mean speed by FMI-Y for each cell or cluster.JY cells or primary B cells . All samples were collected after signing an informed consent, following standard ethical procedures (Helsinki protocol) and with the approval by the local ethics committee (Comité de Protection des Personnes Sud-Ouest et Outremer II).Supplementary InformationVideo legendsVideo 1Video 2Video 3Video 4Video 5Video 6Video 7Video 8


EF) or spatial homogeneity but not both. Small interparticle gaps or sharp nanostructures show enormous EFs but no near-field homogeneity. Meanwhile, approaches using rounded and separated monomers create uniform near-fields with moderate EFs. Here, guided by numerical simulations, we show how arrays of weakly-coupled Ag nanohelices achieve both homogeneous and strong near-field enhancements, reaching even the limit forreproducible detection of individual molecules. The unique near-field distribution of a single nanohelix consists of broad hot-spots, merging with those from neighbouring nanohelices in specific array configurations and generating a wide and uniform detection zone (“hot-volume”). We experimentally assessed these nanostructures via surface-enhanced Raman spectroscopy, obtaining a corresponding EF of ~107 and a relative standard deviation <10%. These values demonstrate arrays of nanohelices as state-of-the-art substrates for reproducible optical detection as well as compelling nanostructures for related fields such as near-field imaging.Reproducible and enhanced optical detection of molecules in low concentrations demands simultaneously intense and homogeneous electric fields acting as robust signal amplifiers. To generate such sophisticated optical near-fields, different plasmonic nanostructures were investigated in recent years. These, however, exhibit either high enhancement factor ( Detecting and identifying individual molecules is the ultimate target of any sensing or diagnostics technique123456789345678912345678937EF, but also extended regions of homogeneous (uniform) and intense near-field strength34The fabrication of metallic nanostructured substrates has considerably evolved with time since the discovery of near-field enhancements at rough silver surfaces638378EF values which are sufficient for detection of molecules in low concentrations, even down to the single-molecule level for the here tested SERS-active molecule6EF of ~107 in our fabricated arrays of metallic nanohelices and a relative standard deviation (RSD) <10%. These values validate these substrates as state-of-the-art substrates for highly enhanced and reproducible SERS detection46Currently, the generation of near-fields satisfying such demanding conditions is attempted by regular arrays of rounded and conveniently spaced individual nanostructures (monomers)31111335116Iinc is ~85, a value which is more than three times higher than the value for nanorodsp. In addition, in this unique near-field distribution, periodic dipoles are formed with an effective length of half a pitch13p/2 (n) positions np; whereas the opposite charge is accumulated on the other side of the nanohelix at positions shifted by p/2: p(n + 1/2). This peculiar charge distribution plays a determining role for the intercoupling strength in arrays of individual metallic nanohelices separated by a distance δ (see discussions below). To assess this, we calculated (Imax/Iincof a nanohelix placed in an array for different ratios δ/(2rw), were rw is the radius of the wire forming the nanohelix.According to far-field studies, the optical response of a nanohelix is governed by localized surface plasmon resonances (LSPR)13141516171819le helix  three el helix 13, inset.  helix 13 due to dtch13p/2 . Thus, slculated  the maxiδ/(2rw), a significant continuous increase in Imax/Iinc is found below δ/(2rw) ~ 0.7, attributed to a strong coupling regime2122δ/(2rw) > 2), Imax/Iinc approaches the value of an individual nanohelix (δ/(2rw), a weakly interacting interparticle coupling regime exists. 3e)·Imax/Iinc from the near-field distributions in While decreasing p/2 (e)·Imax/Iinc, IV, and the total volume existing between neighbouring helices VNH for several values of δ/(2rw). The histogram shows that there is a steady increase of IV/VNH coming from large interparticle distances peaking in the weakly coupled regime for δ/(2rw) ~ 1. Thereafter IV/VNH quickly falls off close to zero below δ/(2rw) ~ 0.7, that is when entering into the strong coupling regime. The combined results in δ/(2rw) ~ 1 is larger than 3 × 105 nm3 and therefore allows for the uniform excitation of even large biomolecules3. Moreover, this value comprises ~8% of the free space in between neighbouring nanohelices; exceeding considerably any reported volume fraction (<0.1% 34567891011δ/(2rw) between 0.8 and 1.1 possess similar Imax/Iinc and normalized hot-volumes IV/VN, a fact suggesting robustness against fabrication tolerances of these nanostructured substratesImportantly, this ability of the weakly coupled regime to generate hot-volumes stems from the exclusive (regular) hot-spot distribution along an individual nanohelix  allowingp/2 , inset. p/2  the ratiEF and RSD at 785 nm incident laser light with the widely used probing molecule3pMA as analyte.To underpin our theoretical considerations on the use of arrays of weakly coupled nanohelices for optical detection of molecules in low concentrations, and thereby confirming their principle applicability, we fabricated3456789pMA (see Methods). Through this, an adsorbed monolayer of these molecules forms on the nanostructured metal surface, as reported in the literature3The choice of the 785 nm laser line is due to the position of the Ag nanohelix LSPR position at ~760 nm . After bpMA covering Ag nanohelix arrays and bulk-powder pMA used as reference. The presence of Ag nanohelices enhances considerably the Raman signal of the monolayer of pMA molecules deposited on their surface. The estimated EF of the present samples is at least 107 as extracted from the two characteristic pMA Raman lines3−1  moleculeshin film . Our obsRSD values of ~10% and ~6% for the two aforementioned Raman lines . We obtained highly repeatable spectra  and estimolecule .δ/(2rw) ~ 1 via weak interparticle plasmon coupling. These volumes comprise up to 8% of the free space inbetween neighbouring nanohelices. By using SERS as a common optical detection technique and without any further optimization, these arrays can reach high EF ~ 107 and RSD ≤ 10%, as exemplified on an array of Ag nanohelices. These values are superior to other (well separated) monomer-based substrates36pMA). In addition, they are comparable to substrates containing narrow hot-spots with interfering filling molecules to achieve a homogeneous near-fieldAltogether, the unique optical near-field of an individual nanohelix allows the creation of large and homogeneous near fields (hot-volumes) in arrays of nanohelices for aspect ratios δ/rw ~ 1 vip ~ 128 nm, diameter D ~ 75 nm, number of turns N ~ 2.8, and wire radius rw ~ 35 nm). The centre-to-centre distance between nanohelices is 150 nm, thus the separation distance between helices is δ ~ 75 nm and the achieved aspect ratio is δ/(2rw) ~ 1. Samples were kept under vacuum after fabrication prior to the Raman measurements.Periodic arrays of regular metallic nanohelices are engineered at the nanoscale by oblique angle deposition on rotating substrates−1 and 521 cm−1, respectively. The micro-Raman resolution is ~2 cm−1. Acquisition time was 1 min, and the laser power was 1 mW. Experimentally, the EF can be determined by the expression3456EF represents the ratio of the SERS signal ISERS to the reference Raman signal, IREF, normalised per molecule, where NSERS and NREF, respectively, are the number of molecules giving rise to the Raman response with SERS and on a reference substrate.Raman spectra acquisition was done at ambient conditions in a Renishaw micro-Raman spectrometer using a ×50 objective lens. The laser wavelength used was 785 nm. The calibration was carried out with the aid of the Rayleigh and Si bands at 0 cmEF determination, we calculate the number of pMA molecules in the bulk-powder reference sample NREF and in our nanostructured substrate NSERS as follows: we have ξ is the focal depth of the laser spot (12 μm for 785 nm wavelength), ρ ~ 6.5 × 1027 m−3 (1.18 g cm−3) is the density of pMA moleculesALASER is the area of the local spot of the laser. However, in our case there is no need to further estimate ALASER since NSERS depends on this variable, too, throughrw is the radius of the wire composing the nanohelix, and −2 is the surface density of absorbed pMA moleculesISERS and IREF from the experimental Raman pMA peaks at ~1080 cm−1 and ~1180 cm−1, we find an overall EF ~ 0.9 × 107. We find the same EF magnitude also when using pMA on a thin film of Ag as reference (Regarding the eference .−5 M) of pMA. Through this we achieved a monolayer of these molecules attached to the nanohelices’ surface due to the common chemisorption process3pMA to silver.We exposed the Ag nanohelices for ~12 hours to an aqueous solution (102 and Si base wafer); no differences were observed.We simulate the near-field intensity of individual and arrays of nanohelices using the commercial finite element modelling (FEM) package, COMSOL . In all δ. Finally, the optical properties of the nanohelices are defined through the real and imaginary components of the refractive indexLight is incident along the negative direction in the z axis. The helices used have a pitch of 130 nm, a diameter of 75 nm and a diameter of the helical wire of 70 nm. All these values are close to those of the experimentally fabricated nanohelices. The scattered field is absorbed by perfectly matched layers (PML) regions. For all  of nanohelices, the simulation domain is enclosed by five PMLs. Five elements were needed across the PML region to accurately absorb scattered field with less than 1% back reflection. In the case of arrays of nanohelices, we estimated the intercoupling effect in helical arrays by taking into account the contribution from the four nearest neighbours at different particle separation distances How to cite this article: Caridad, J. M. et al. Hot-Volumes as Uniform and Reproducible SERS-Detection Enhancers in Weakly-Coupled Metallic Nanohelices. Sci. Rep.7, 45548; doi: 10.1038/srep45548 (2017).Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


The Aβ+ group exhibited significantly higher ReHo in the fusiform gyrus and lower ReHo in the precuneus compared with the Aβ- group. We found significant negative correlations between global Aβ retention and ReHo in the precuneus and medial prefrontal cortex and positive correlations between global Aβ retention and ReHo in the bilateral lingual gyrus, left fusiform gyrus, and right middle temporal gyrus in the Aβ+ group. Our findings suggest that regional functional synchronization might have distinctive association patterns with Aβ retention in the cognitively normal older adults. These findings can enrich the functional characterization of early stages of disease progression in AD.Previous studies have shown aberrant functional connectivity in preclinical Alzheimer’s disease (AD). However, the effects of beta-amyloid (Aβ) retention on regional functional synchronization in cognitively normal older adults still remain unclear. The aim of this study was to explore the distinctive association pattern between Aβ retention and regional functional synchronization in cognitively normal older adults. Sixty-one older adults with normal cognition underwent functional magnetic resonance imaging and regional functional synchronizations were quantified using regional homogeneity (ReHo). Subjects were dichotomized using  In the cognitively normal older adults with Aβ deposition, there is an increased risk of longitudinal cognitive decline and conversion to the symptomatic AD phenotype10. However, Aβ deposition has been suggested to be necessary, but not sufficient, for the development of AD pathogenesis. Indeed, additional events must occur before cognitive decline and the progression to AD11. In this regard, attention has been focused on mediating factors such as functional brain changes occurring in between Aβ deposition and cognitive decline.Beta-amyloid (Aβ) deposition plays an important role in the pathogenesis of Alzheimer’s disease (AD). Approximately one third of cognitively normal elderly adults are reported to have some evidence of Aβ deposition13. Several prior studies have shown that aberrant functional connectivity (FC) within intrinsic functional networks such as the default mode network (DMN) and central executive network reflected the progression of pathology in the clinical spectrum of AD15. Although FC abnormalities within the intrinsic functional networks can show integrative pathological changes between two or more discrete brain regions, they cannot identify the particular region responsible for clinical symptoms. To overcome these methodological limitations of FC, regional homogeneity (ReHo) has been developed to evaluate regional resting state brain activity. ReHo is evaluated by Kendall’s coefficient of concordance (KCC) to determine the degree to which the time series of a given voxel is synchronized with its neighbors17. ReHo is based on the assumption that when brain activity is initiated, it is activated in the form of a cluster rather than a single voxel. Furthermore, ReHo has been reported to reflect intra-regional synchronization, to reveal unpredicted regions and to be more sensitive to the default mode network than other parameters including FC18. In these regards, several prior studies have evaluated ReHo changes along the AD continuum. In individuals with mild cognitive impairment (MCI), ReHo was decreased in the medial prefrontal cortex, bilateral posterior cingulate cortex and precuneus, but increased in the left inferior parietal lobule, right fusiform gyrus and bilateral putamen compared with cognitively normal elderly20. In AD patients, ReHo was decreased in the medial prefrontal cortex, bilateral posterior cingulate cortex, and precuneus, but increased in the bilateral cuneus, left lingual gyrus, and right fusiform gyrus21. These regions are similar to the regions of the default mode network (DMN) and the compensatory region. Moreover, the value of ReHo showed a positive correlation with episodic memory functions21. In this regard, ReHo has been postulated to act as a noninvasive biomarker reflecting the progression of AD19. However, only a few studies have been conducted and were limited by a small number of subjects and lack of identification of Aβ deposition. Although numerous studies have reported the detrimental effect of Aβ deposition on bimodal FC and glucose metabolism in cognitively normal older adults, no study has examined the impact of Aβ deposition on ReHo in cognitively normal elderly23.Resting state functional MRI (fMRI) reveals spontaneous neuronal activity of the human brain in the resting state and measures useful parameters in evaluating the elderly who have difficulty performing tasksThe aim of our study was to determine the impact of Aβ deposition on ReHo in cognitively normal older adults. To distinguish the effects of normal aging from the effects of older subjects at risk of AD, we examined group differences of ReHo between cognitively normal older adults with (Aβ+) and without (Aβ−) Aβ deposition. Moreover, we attempted to explore the association between Aβ deposition and ReHo in the Aβ+ group and evaluated the relationships between ReHo and memory performance in the Aβ+ group versus the Aβ− group. Finally, we explored the sensitivity and specificity of the mean ReHo values in discriminating between the Aβ+ and Aβ− groups.19, we hypothesized that ReHo of the Aβ+ group would be substantially decreased when compared with that of Aβ− group. Furthermore, we expected that Aβ deposition would be negatively associated with ReHo in the Aβ+ group. In addition, we hypothesized that there would be significant differences between the Aβ+ and Aβ− groups in the association between episodic memory performance and ReHo maps. Finally, we postulated that mean ReHo values would have significant sensitivity and specificity for discriminating between the Aβ+ and Aβ− groups.As previous studies have reported a decreased ReHo index in regions of the DMN during the progression of ADTable The Aβ+ group showed significantly higher retention of Aβ in the anterior cingulate cortex, precuneus, middle frontal cortex and lateral temporal cortex compared to the Aβ− group Fig. , false dThe mean ReHo maps within each group are shown in Fig. 18F)(FBB) retention and ReHo values in the Aβ+ group alone. In the Aβ+ group, ReHo in the left and right superior medial frontal gyrus, precuneus, left orbitofrontal cortex and left angular gyrus showed a significant negative correlation with the global FBB retention (AlphaSim corrected P < 0.001). In addition, ReHo in the left and right lingual gyrus, left fusiform gyrus and right middle temporal gyrus was positively correlated with the global FBB retention in the Aβ+ group (AlphaSim corrected P < 0.001).Figure Figure To the best of our knowledge, this is the first study to determine the impact of Aβ burden on regional functional synchronization in cognitively normal older adults. A key strength of the present study was the measurement of Aβ retention by amyloid positron emission tomography (PET) for differentiating cognitively normal older adults at risk of AD from normal controls. Hence, we could conduct a more thorough investigation on the relationships between the degree of Aβ retention and the ReHo value as well as differences in ReHo between cognitively normal subjects at risk of AD and healthy controls to clarify changes in intra-regional brain activity during the earliest phase of AD.24, which is in agreement with a previous study which showed a similar distribution of higher ReHo in cognitively normal older adults and AD subjects21. To date, several prior works showed that topographical distributions of Aβ retention appeared to overlap the regions within the DMN, which has a higher basal metabolic activity, compared with the other brain regions25. Furthermore, the positive relationship between Aβ retention and intrinsic FCs has been shown at the global network level from the earliest stage of AD26. In this regard, Aβ retention has been suggested to be accelerated28 and preceded30 by increased metabolism, intrinsic activity and connectivity. Collectively, we could infer that the pattern of ReHo reflecting intra-regional brain activity would be similar to that of resting metabolic activity in the DMN, with overlapped regions of Aβ retention at the earliest stage of AD.The current study found that regions with higher Aβ retention in the Aβ+ group overlapped with regions with higher ReHo in both groups. These overlapping regions were consistent with the components of the DMN including the anterior cingulate cortex and precuneus21. However, the findings of our study do not support earlier investigations using fluorodeoxyglucose (FDG)-PET, which have demonstrated non-significant differences of regional metabolism between cognitively normal subjects with and without Aβ retention31. Furthermore, the current study found a significant negative correlation between the global Aβ retention and the ReHo within the DMN regions including precuneus in the Aβ+ group. This is also in accordance with earlier observations, which showed that the negative impact of Aβ retention on FCs and local metabolic activity in the posterior DMN of cognitively normal adults with Aβ deposition34. However, this finding appears to be contrary to aforementioned results which have suggested that heightened intrinsic brain activity may induce amyloid retention. A possible explanation for this discrepancy may be that Aβ retention has the driving force of reducing the metabolic activity only after extensive Aβ deposition induced by higher basal metabolism35. In addition, the reduction of intra-regional metabolic activity has been demonstrated to initiate from the earliest phase of AD and to peak at the prodromal stage of AD26. Therefore, an intra-regional brain activity measured by the cross-sectional analyses in cognitively normal subjects at risk of AD could be affected from the extent to which intra-regional brain activity is reduced. Therefore, inconsistent differences of intra-regional brain activity between cognitively normal subjects with and without Aβ retention might be interpreted with the aforementioned proposition.We also found that the Aβ+ group showed lower ReHo in the left precuneus compared with the Aβ− group. This result is in line with previous studies that showed lower ReHo in the posterior cingulate cortex/precuneus in MCI and AD compared with control group21. Moreover, the higher intra-regional activity of the fusiform gyrus was suggested to be compensatory for the progression of AD pathogenesis21. This is also in accordance with earlier observations, which showed the positive correlation between the ReHo of compensatory regions (including the fusiform gyrus) and individual intelligence36. This finding is also exemplified in the previous research, which have shown that increased Aβ retention is associated with increased functional connectivity of lateral temporal cortices and is suggested to be compensatory32.We found that the Aβ+ group showed higher ReHo in the left fusiform gyrus compared with the Aβ- group. Furthermore, the present study found a significant positive correlation between Aβ retention and ReHo in the bilateral lingual gyrus, left fusiform gyrus and middle temporal gyrus in the Aβ+ group. These results are concordant with the findings of previous studies which showed higher ReHo in the fusiform gyrus in MCI and AD subjects compared to healthy controls19. Taken together, these results strengthen the idea that the mean ReHo value might be sensitive to earlier changes in spontaneous brain activity in response to Aβ retention. However, further longitudinal study with larger sample size would be needed to confirm the ReHo as a potential functional biomarker for predicting the risk of AD.Another important finding was that the mean ReHo values of the left precuneus showed higher AUC, sensitivity and specificity in discriminating the Aβ+ group from the Aβ− group. In previous research, the mean ReHo values could discriminate subjects based on the stage of cognitive impairment  at a rate that was 71.4% correct21. The precuneus/posterior cingulate cortex has been documented to activate during memory retrieval37, to network with the medial temporal lobe and to engage in memory function in the early phase of AD25. In addition, these results are in line with our previous study showing group × episodic memory interactions in the posterior DMN FC and positive correlations between posterior DMN FC and episodic memory function scores in the cognitively normal older adults with Aβ retention34. Therefore, our results with positive correlation between the precuneus ReHo and memory retrieval suggest the downstream effect of the Aβ deposition on the left precuneus ReHo, where lower functional synchronization is associated with poorer episodic memory38. However, these simple association patterns are not sufficient to clarify the role of the ReHo linking Aβ retention and cognitive impairments, which are essential characteristics of AD4. In these regards, we conducted a mediation analysis to clarify whether the left precuneus ReHo mediated the link between Aβ deposition and episodic memory impairments. Interestingly, the mediation analysis results showed that the left precuneus ReHo completely mediated the link between Aβ retention and cognitive impairments. These results may help to explain the inconsistent results of association between Aβ retention and cognitive impairments in the cognitively normal older adults34. In addition, our results also expands several prior works showing mediation effects of the DMN FCs and brain glucose metabolism between Aβ retention and cognitive impairments in the cognitively normal40 and the prodromal AD subjects41. However, further longitudinal prospective studies will be necessary to confirm mediation effects of the ReHo between Aβ retention and cognitive impairments on the trajectories of AD.In the current study, we also found significant group × memory retrieval interactions in the left precuneus. Furthermore, both groups showed a positive correlation between the ReHo and memory retrieval in the left precuneus. These findings are consistent with that of previous study, which have shown the positive relationship between ReHo of the posterior cingulate cortex/precuneus and scores of mini mental status examination in the AD patient group44. Therefore, further longitudinal, controlled research should be conducted in order to determine the causal effect of Aβ retention on ReHo, or vice versa.The major limitation of this study is the use of a cross-sectional design. The cross-sectional design precludes our ability to make causal inferences; however, it allows us to generate hypotheses for future studies. Therefore, these cross-sectional findings require validation in prospective studies. Another limitation includes the lack of examination of the apolipoprotein epsilon4 (APOE4) allele and tau pathology which has been reported to be associated with brain activity in the DMN in cognitively normal younger and older adultsIn summary, this study identified alterations in ReHo, differential patterns of association between Aβ deposition and ReHo, the relationship between memory retrieval and ReHo in cognitively normal older adults with Aβ retention. Moreover, the features of ReHo in discriminating cognitively normal older subjects at risk of AD from those in normal aging process. This combination of findings provides some support for the value of intra-regional brain activity for understanding the earliest pathogenesis of AD. However, more longitudinal research on this topic needs to be undertaken in conjunction with other parameters for clarifying the role of ReHo in the progression of AD.45. Subjects with any psychiatric, neurological and unstable medical conditions were excluded. The cognitive testing battery included the following domains: memory, visuospatial construction, language, attention and executive functions. Details on the specific tests used and the reviewing process are described in the Supplementary material. The study was conducted in accordance with the ethical and safety guidelines set forth by the local Institutional Review Board of the Catholic University of Korea and written informed consent was obtained from all study subjects. The local Institutional Review Board of the Catholic University of Korea approved this study (No. VC15EISI0044) following the principles set forth by the Declaration of Helsinki.Sixty one elderly subjects with normal cognition were included in this study. They were recruited from the normal control volunteers of the Catholic Dementia Brain Imaging Database, which holds brain scans of outpatients and inpatients at the Department of Geriatric Psychiatry, Saint Vincent Hospital, The Catholic University of Korea from 2010 to 2016. The inclusion criteria of the subjects were as follows: (i) subjects aged ≥60 years; (ii) Mini-Mental Status Examination score ≥27; and (iii) Clinical Dementia Rating = 046. Each individual participant’s MRI was utilized for co-registration and defining the ROI and for correcting partial volume effects from expanding cerebrospinal spaces accompanying cerebral atrophy48. Analysis of the FBB PET data utilized a standardized uptake value ratio (SUVR) 90 min post-injection, using the cerebellar cortex region of interest as the reference. Global Aβ burden was expressed as the average SUVR of the mean for the following 5 cortical ROIs: frontal, superior parietal, lateral temporal, and anterior and posterior cingulate cortex/precuneus as described in previous study48.FBB was produced and FBB-PET data were collected and analyzed as previously described49. PET image preprocessing and voxel wise FBB PET analysis processes are described in detail in the Supplementary material.The FBB PET data were acquired within 4 weeks of clinical screening and cognitive function test. We used a cut-off for ‘high’ or ‘low’ neocortical SUVR of 1.4, consistent with cut-off values used in previous FBB-PET study3. Resting state functional images were collected using a T2* weighting gradient echo sequence with TR = 2,490 ms, TE = 30 ms, matrix = 128 × 128 × 29, and voxel size = 2 × 2 × 3 mm3. One hundred and fifty volumes were acquired over 5 minutes with the instruction “keep your eyes closed and think of nothing in particular”.Imaging data were collected at the Department of Radiology, St Vincent’s Hospital, The Catholic University of Korea, using a 3T Siemens Verio machine and eight channel Siemens head coil. The parameters used for the T1-weighted volumetric magnetization-prepared rapid gradient echo scan sequences were TE = 2.5 ms, TR = 1,900 ms, inversion time = 900 ms, FOV = 250 mm, matrix = 256 × 256, and voxel size = 1.0 × 1.0 × 1.0 mm50 which is based on Statistical Parametric Mapping  to preprocess the fMRI images. Slice timing and realignment for motion corrections were performed on the images. Subjects with excessive head motion (cumulative translation or rotation > 2mm or 2°) were excluded. To prevent group-related differences from micro-head motion, framewise displacement (FD) was compared between the groups. Mean FD scores were not different between the groups  and further used as covariates in group comparisons. In spatial normalization, the International Consortium for Brain Mapping (ICBM) template was applied (resampling voxel size = 3mm × 3mm × 3mm) which was fitted to the ‘East Asian brain’.We used the Data Processing Assistant for Resting-State fMRI (DPARSF)50. Above all, linear trends were removed from the functional images. After this, the data were filtered with a temporal band-pass of 0.01–0.08 Hz. This filtering reduces low- frequency drift as well as physiological high-frequency respiratory and cardiac noise12. Next, ReHo maps of all participants were made by a general routine using DPARSF. Briefly, we set the basic cube to calculate the KCC by 3mm × 3mm × 3mm voxels. Therefore, the KCC value of the central voxel in the cube was calculated by referring to the temporal sequences of its neighboring 26 voxels. The calculated value was assigned as the ReHo value of the central voxel. This calculation was repeated for all the voxels throughout the brain; subsequently, an unsmoothed ReHo map was drawn. This raw ReHo map was smoothed by 6mm of full width at half maximum (FWHM). This smoothing is appropriate for the cluster-level analysis which is described in the next section52.We further processed our functional data to make them fit for ReHo analysis through the Data Processing Assistant for Resting-State fMRI2 test were used to assess potential differences between the Aβ+ and the Aβ− groups for all demographic and clinical variables and SUVR values. All statistical analyses used a two-tailed level of 0.05 for defining statistical significance. The general linear model (GLM) was used for measuring the within and between group differences of the ReHo maps. To examine the relationships between Aβ deposition and ReHo in the Aβ+ group, the global mean SUVR value from the 5 ROIs were correlated with the voxel-wise ReHo maps of the brain using GLM.Statistical analyses for demographic data were performed with the Statistical Package for Social Sciences software . The independent t-test and the χMean ReHo values from brain regions with significant group differences were used further ROIs analysis. We performed binary logistic regression with ROC analysis to evaluate the sensitivity and specificity of the mean ReHo values from the ROIs to discriminate the Aβ+ group from the Aβ− group. In addition, the GLM with ReHo as the dependent variable Word List Recall (CERAD WLR) scores and group as the independent variables were performed, as well as analysis on the interaction (episodic memory × group). We controlled for the effect of age, education and gender in all GLM analysis used.Multiple corrections were performed using cluster-extent correction (AlphaSim) as implemented through DPABI, and the parameters were set as follows: individual voxel threshold p < 0.001, number of Monte Carlo simulations = 1000, and p = 0.001 as the effective threshold for cluster-extent correction.53 controlled for age, sex, and education level.Furthermore, to address the question of whether the regional functional synchronization mediated the association between Aβ retention and episodic memory performances in the Aβ+ group, a mediation analysis was performed using the PROCESS macroThe datasets generated and analyzed during the current study are available from the corresponding author on reasonable request.supplemental materials


Pharmaceutical products, particularly antibiotics, due to their cumulative characteristics, undesirable effects and creating drug resistances, as inevitably pollutants, poses a major concern in environmental issues. In recent years, advanced oxidation processes (AOP) have been considerably used for degradation of new and emerging pollutants such as residual medications and resistant compounds in water and wastewater. Present investigation evaluates the removal of metronidazole from aqueous solution by electro coagulation and photoelectrophenton processes. The data will be informative for environmental agencies, pharmaceutical companies and wastewater treatment companies for choosing it as a practical oxidation advance process for treatment of water polluted by resistant material (drugs and pesticides). Specifications TableValue of the dataThe data may be useful for future researches that aimed in pharmacy wastewater treatment.This data allows wastewater treatment plants managers and engineers to extend the practical usage of phenton process.Our data showed that photo-electro-phenton process remove antibiotics from wastewater; an interesting issue for environmentalists who concerned about pharmacy wastewater treatment.12O2 concentration, UV irradiation, and metronidazole concentration on degradation of metronidazole are presented in This brief dataset describes the use of electro coagulation and photo-electro-phenton process for removing an antibiotic from synthetic wastewater. 2In this study, a Plexiglas reactor with working volume of 1 l was used. The rectangular reactor was equipped with two electrodes (iron anode with dimensions (120 × 5 × 120 mm)) and a graphite cathode with dimensions (150 × 120 × 150 mm) and a 5 cm distance to each other. The distance between the electrodes and the reactor wall was 1 cm. An air pump and a ceramic diffuser used for aeration


Outer primary feathers were used from 661 after-second-year (ASY) male blackbirds collected in Minnesota, Montana, North Dakota and South Dakota (spring collection), and Arkansas, Louisiana, Mississippi, Missouri and Texas (winter collection). The best-fit model indicated that the combination of feather δ2H, δ13C and δ15N best predicted the state of sample collections and thus supported the use of this approach for tracing molt origins in Red-winged Blackbirds. When considering only birds collected in spring, 56% of birds were classified to their collection state on the basis of δ2H and δ13C alone. We then developed feather isoscapes for δ13C based upon these data and for δ2H based upon continental patterns of δ2H in precipitation. We used 81 birds collected at the ten independent sites for model validation. The spatially-explicit assignment of these 81 birds to the δ2H isoscape resulted in relatively high rates (~77%) of accurate assignment to collection states. We also modeled the spatial extent of C3  and C4  agricultural crops grown throughout the Upper Midwest and Mid-South United States to predict the relative use of C3- versus C4-based foodwebs among sampled blackbirds. Estimates of C3 inputs to diet ranged from 50% in Arkansas to 27% in Minnesota. As a novel contribution to blackbird conservation and management, we demonstrate how such feather isoscapes can be used to predict the molt origin and interstate movements of migratory blackbirds for subsequent investigations of breeding biology (e.g. sex-specific philopatry), agricultural depredation, feeding ecology, physiology of migration and sensitivity to environmental change.We analyzed stable-hydrogen ( Agelaius phoeniceus) have experienced declines since 1965 . Th. Thδ2H vδ2Hf value against the isoscape predicted (as derived above) δ2H value for a given pixel within the isoscape. We parameterized the normal probability density function by treating isoscape predicted δ2Hf values (within every pixel) as the means against which δ2Hf was compared [‰), spatially-explicit prediction errors of a previous isoscape [‰. Applying the normal probability density function resulted in one map of posterior probabilities for each bird in the sample.We conducted assignments to origin by applying normal probability density functions to provide a spatially-explicit assessment of the likelihood that a given feather was grown at a given location within the isoscape. This was accomplished by comparing the observed compared , 30. In compared . These visoscape  and unceδ2Hf was less than (north) or greater (south) than the value predicted from the calibrated isoscape at the collection site. Finally, we applied our algorithm to assign the 81 birds sampled at ten independent sites to validate our assignment accuracy.In order to assign each bird to likely origins, we used a 2:1 odds ratio of being “correct” versus “incorrect” by selecting the upper 67% of the probability densities and recoded these as “likely” origins , 33. If δ13C, δ15N) of feathers between birds classified as resident, migrants from north of the collection sites, and migrants from south of the collection sites based on the designations derived above. We did not include δ2H values in this multivariate comparison, because our geospatial assignment approach would result in defacto differences. We conducted perMANOVA based on 999 permutations of Euclidean distances using the vegan R package [δ13C χ2 = 15.06, df = 2, p < 0.001; δ15N χ2 = 12.82, df = 2, p < 0.001) required for parametric MANOVA tests. Geospatial assignments to origin were conducted using customized functions for the ‘raster’ package [We used a permutation-based multivariate analysis of variance (perMANOVA)  to compa package . We used package . All ana package .δ2H (˗153.0 to ˗20.3 ‰), δ13C (˗32.2 to ˗10.1 ‰) and δ15N (4.7 to 16.7 ‰) values in feathers of blackbirds  and longitude  of collection sites. Feather δ13C values were also inversely related to latitude  and positively related to longitude . Values of δ15N were inversely related to both latitude  and longitude of collection sites . We observed the greatest differences between states in δ2Hf values. Non-overlapping, 95% confidence intervals suggested that δ2Hf values were different for feathers collected in Texas, Minnesota, South Dakota, North Dakota and Montana . Thus, the latitude  and longitude  of collection sites were related to the combination of δ2H, δ13C and δ15N values for blackbird feathers.When considering only birds collected in spring, 56% of birds were classified to their collection state on the basis of 3C alone . Althougδ13C) linear mixing model. However, because we did not measure δ13C values of crops directly at each collection site, and because there can be a considerable isotopic range across plants in each of these categories [δ13C values. In general, the feather δ13C data conformed well to expectations of a broad C3 to C4 isotopic range reflecting the isotopic difference between C3 and C4 plants, or about -27 ‰ to -12 ‰, respectively [th and 95th percentiles of the distributions and, if these conformed well to the expected 15 ‰ C3 to C4 range of feather δ13C values for each population (i.e. that range expected between a pure C3 and a pure C4 diet) [th percentile or above the 95th percentile were considered to have 100% C3 and C4 diets, respectively. We determined a broad range of C4 dietary inputs among individuals within populations , single-isotope  , we assoulations .δ2Hf values from known-origin feather samples against the predicted values resulted in the isotropic stable variogram model being heavily favored, receiving 95% of the support based on AICc model weights. The selected model had a lag distance of ~80.6 km, a range of ~718 km, a nugget of 132.01, and a partial sill of 380.20. The resulting isoscape predicted an isotopic gradient that was most enriched in 2H  in the southeastern portion of our study area, and most depleted in 2H in the northwest , a slope for δ2H  and the average proportion of C4 crops in the county in which the bird was collected . This model explained ~11% of the variation in δ13C, and suggests that birds collected in regions of the study area that were most depleted in 2H  were also the most depleted in 13C , and a slope that departed substantially from 1 .Based on AICameters” , the topd in 13C , and thad county . While tδ2Hf isoscape resulted in 62 individuals from our validation sample of 81 birds (~77%) being accurately assigned to the site at which they were collected in spring. We therefore applied this model to assign birds collected in winter to their putative molt origins. We collected 372 blackbirds on the wintering grounds, 215 of which were assigned to likely origins that overlapped their sampling locale and were thus considered residents. The remaining 157 wintering samples were considered migrants, 32 of which had putative origins north of their collection sites (n = 1) and Alaska  of blackbirds feathers . Graphical examination of the data suggests that the multivariate difference was primarily due to variation in feather δ13C values, with migrants from the north tending to be substantially more depleted in 13C than either residents or migrants from south of their collection sites  and residents  explained approximately 10.1% of the variation in the isotopic composition  of accurate assignment to collection states. Eighty percent of their 64 samples was classified to their mid-May–mid-July collection sites using δ2H and δ13C and values [δ2H values were used, indicating that δ13C provided a useful additional means of delineating origins of blackbirds [It was previously suggested that the feather isotope approach could be used to investigate the origins of Red-winged Blackbirds potentially associated with agricultural crop depredation throughout the continental Midwest . When cod values . This peackbirds .Our extensive analyses of primary feathers from Red-winged Blackbirds collected in spring and winter indicated a broad isotopic range generally consistent with latitudinal variation in origins and use of diets based on carbon spanning a range of C3 to C4 photosynthetic pathways. These attributes predisposed this species as a good candidate for the forensic evaluation of both geographical origin and use of agricultural crops during the post-breeding molt. Our results confirm the utility of the isotope approach to investigating movements and ecology of blackbirds and other species associated with agriculture in North America and elsewhere. However, the complex nature of agricultural landscapes, especially those involving the juxtaposition of C3 and C4 crops combined with the possibility that blackbirds may disperse from the breeding locations to other areas in order to molt, makes the precise modeling of blackbird origins extremely challenging. Nonetheless, we have demonstrated that the combination of stable isotope measurements of feathers from blackbirds collected on their wintering grounds can provide important information on the structure of wintering populations, migratory connectivity and ultimate indices of crop and other dietary sources.δ2H, δ13C and δ15N values in feathers of blackbirds, our stepwise discriminant analysis indicated that the inclusion of each isotope value provided the best-fit model for the classification of collection states. Thus, combined analyses of δ2H, δ13C and δ15N values can be used for subsequent investigations of interstate, national and international movements, migration, and spring and winter habits of migratory birds and other wildlife. Moreover, conservation and management efforts can now be planned and implemented with an understanding of the subpopulations, movements and diets of wildlife at appropriate spatial scales.Although we observed a considerable range in δ13C marker of blackbird use of the C4 crops. However, non-agricultural  diets would presumably be mostly C3 and so the relative amount of C3 crops to diets is less readily inferred from isotopic measurements alone. Our estimate of the maximum use of C4 crops during molt was 50% for wintering blackbirds collected in Arkansas. This was followed closely by birds from South Dakota, Missouri and Texas. Relatively low (~27%) C4 inputs were estimated for diets of blackbirds collected in Missouri, Minnesota and North Dakota. Additional analyses are planned for parsing the post-molt dispersal of blackbirds collected in spring and winter relative to C3- and C4-based foodwebs.The large isotopic difference between C3  and C4  crops provided a convenient 15N values can be modeled based on climate variables such as rainfall amounts that are known to influence plant metabolism [15N values in aquatic organisms with human-dominated landscapes under heavy nitrogen loading [15N values in terrestrial organisms with arid regions [15N values, tracing organisms to δ15N isoscapes requires a good understanding of isotopic discrimination. The relative constant feather δ15N values we found suggest that the agricultural landuse across regions approached maximum N fertilizer inputs and levels of intensity. This, in turn, resulted in low discrimination power for this isotope with the exception of Texas.Stable-nitrogen isotope measurements provided the least information in our samples with most populations exhibiting mean values in the range of 10‰. The exception was Texas at 12‰ and this may reflect the more arid conditions in that region in 2013. Plant δtabolism  but thistabolism . General loading  and high regions . However regions . Additioδ2Hf values. As expected, the selected model predicted greatest δ2Hf values in the southeast and least δ2Hf in the northwest portion of our study area. Thus, as has been demonstrated in numerous other studies [2Hf analyses can provide reliable inferences regarding the general latitudinal origin of feathers sampled from blackbirds and this can be further refined to state delineations in some cases. The most likely complicating factor with this isotope is the potential use of groundwater for irrigation. That practice can result in overall enrichment in 2H compared to continental models of growing-season average precipitation and may account for the enriched feather isotope values seen in the Texas sample [2H and 13C may be relatively enriched in the foodweb. Birds that molted along the coast may have moved inland to take advantage of rice fields, consistent with previously described daily movements of ~ 75–85 km from coastal roosts to inland rice fields [δ2H and δ13C due to agricultural practices alone . Evaluation of these alternative hypotheses will require further collection of calibration samples during the breeding/molting period along the southern range edge, and/or validation using telemetry based methods.Stable-hydrogen isotopes from known-origin feather samples were strongly associated with predicted  studies , 44, δ2Hs sample . Our obse fields . Alternaδ2H measurements) and the relative use of C4 crops (using δ13C measurements) during molt. On wintering grounds, examining the proportion of local versus immigrant birds in wintering flocks has considerable potential for devising more refined strategies for managing blackbirds in an agricultural environment. In addition, there is potential to examine how flock composition may change through time and in response to management prescriptions on the breeding and wintering grounds.Our study provides a foundation for future research into using stable isotope tracers to investigate the ecology of birds associated with agro-ecosystems. In our case, more research into the appropriate scales of model development using state crop statistics is warranted. Currently, it is not clear if isoscape modeling can be refined to the scale needed to improve assignment at the state level. Blackbirds from single sites showed a considerable range in stable isotope values. Thus, individuals may move considerable distance from their breeding sites to molt  and this can complicate ground-truthing or calibration studies. Currently, the most parsimonious use of the isotope data is to assign molting latitude  feather isotopes for red-winged blackbirds associated with agro-ecosystems. Figshare 2016; https://dx.doi.org/10.6084/m9.figshare.3860772.(XLS)Click here for additional data file.


Due to the high sensitivity of FWM to excitons, tied to their longer coherence decay times than unbound electron- hole pairs, we show that the exciton resonance energies can be directly observed from the nonlinear optical spectra. Our results indicate low-temperature binding energies of 13 meV (29 meV) for the free (defect-bound) exciton, with the 16 meV localization energy for excitons attributed to binding to point defects. Our findings shed light on the wide range of binding energies (2–55 meV) reported in recent years.Solar cells incorporating organic-inorganic perovskite, which may be fabricated using low-cost solution-based processing, have witnessed a dramatic rise in efficiencies yet their fundamental photophysical properties are not well understood. The exciton binding energy, central to the charge collection process, has been the subject of considerable controversy due to subtleties in extracting it from conventional linear spectroscopy techniques due to strong broadening tied to disorder. Here we report the simultaneous observation of free and defect-bound excitons in CH Eb), which determines the primary mechanism of carrier transport during device operation. Unlike inorganic solar cell materials such as III-V semiconductors, in which sharp exciton resonances greatly simplify extraction of Eb, the solution-processed organometal halide perovskites possess substantial broadening due to defects26Eb from absorption or ellipsometry techniques56Eb from magneto absorption techniques is also complicated by the strong broadening in the organometal halide perovskites, resulting in the need to use large magnetic fields (~20 Tesla)93NH3PbI3, the value of Eb has been quite controversial, with reports ranging from 2 meV to 55 meV234789101112131415161718The efficiency of solar cells using organometal halide perovskite semiconductor absorber layers has skyrocketed during the past few years, now reaching a level competitive with existing solar cell technologies2345678910111213141516171819202122233NH3PbI3. In this technique, two coherent ultrafast laser pulses described by electric fields T2)T2 times than delocalized free carrier and band tail transitions29Ga antisite defects preventing the observation of any signature of the exciton in linear absorption, as well as the exciton resonance at the spin-orbit split-off band gap in InPHere we apply femtosecond four-wave mixing spectroscopy (FWM) to probe excitonic resonances in CH2 −  see . Measure3NH3PbI3 thin films, which we attribute to the free exciton transition and a defect-bound exciton transition. The simultaneous observation of both free and localized excitons using this absorption-based nonlinear optical technique shows that both types of exciton can contribute to absorption during operation of a solar cell. At 10 K, the excitonic resonances we observe occur at 1.629 eV and 1.613 eV. The 16 meV energy separation is attributed to the binding energy of excitons to point defects within the perovskite film3NH3PbIx3−Clx using photoluminescence and THz techniques91323478910111213141516171819Our experiments reveal two discrete resonances below the band gap of the CH3NH3PbI3 sample at 10 K is shown in The four-wave mixing signal measured on the CHμJ/cm2. The existence of a separate contribution to the FWM signal below the band gap is already apparent at early delays from the shape of the contours in the results for excitation at 1.653 eV in 24203NH3PbI3 film.Further insight into the transitions contributing to the FWM response may be gained by performing measurements for which the laser pulses used to excite the sample are tuned relative to the band gap energy. The results of these experiments are shown in The variation of the magnitude of the FWM signal as the laser pulses are tuned indicates that the resonance feature at 1.613 eV is not the only discrete resonance below the band gap. A stronger resonance occurs at higher energies, as shown in 3NH3PbI3234789101112131415161718Eb in recent years. The uncertainty in determining the dielectric constant, including both the choice of appropriate spectral range1317et al., the dielectric constant was taken as a fitting variable in modeling the linear absorption spectrum, yielding a free exciton binding energy of 13 meV at 80 K, in agreement with the value reported here.The binding energy of 13 meV we measured for the free exciton is in line with the value of 16 meV found from recent magneto-optical measurements at 2 K973NH3PbI3 film. The energy levels tied to a variety of point defects were calculated using density functional theory techniques by Yin et al.i may be responsible for exciton localization at low temperatures in our sample. The small 16 meV binding energy of these defects is consistent with a similar coherence decay time observed for the free and defect-bound excitons in our experiments , which was maintained at 70 °C throughout the deposition, was first stirred for 10 hours and then passed through a 0.45 μm pore PTFE filter into a clean glass vial heated to 70 °C. 300 μL of the solution was cast onto a room temperature, stationary sapphire substrate, which was subsequently spun at 5000 rpm for 60 seconds. Residual solvent was removed by immediately transferring the substrate to a 70 °C hotplate for 15 minutes. 300 μL of methylammonium iodide solution in isopropanol was deposited onto the spinning lead-iodide film . The films were annealed at 100 °C for 2 hours under an argon atmosphere. X-ray diffraction studies were carried out on a companion sample grown with the same recipe, and indicated no residual lead iodide. Prior to optical measurements, the completed samples were transferred into a portable liquid helium flow-through optical cryostat within an argon glovebox and sealed prior to removal of the cryostat to air. The cryostat was then evacuated for low-temperature optical studies under vacuum. The band gap was extracted from linear absorption measured by a CARY UV-VIS spectrometer following the findings of EvenA modified sequential deposition procedure was used to prepare the thin film CHUltrashort pulses are generated by a Ti:Sapphire mode-locked laser source, pumped by a solid state Nd:Vanadate laser, and passed through a group-velocity dispersion compensator prior to entering the four-wave mixing setup. A beam splitter produces two pulses with electric fields How to cite this article: March, S. A. et al. Simultaneous observation of free and defect-bound excitons in CH3NH3PbI3 using four-wave mixing spectroscopy. Sci. Rep.6, 39139; doi: 10.1038/srep39139 (2016).Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


Using mussel haemocytes, a well-known model system to assess nanomaterial toxicity, we simultaneously assessed zinc accumulation and multiple cellular response endpoints. We found that intracellular zinc uptake was strongly enhanced by inulin coating, in comparison to the uncoated nanoparticles, while no significant effects on cell death, cell viability, mitochondrial membrane integrity, production of reactive oxygen species or lysosome abundance were observed at concentrations up to 20 ppm. Since no significant increments in toxicity were observed, the coated nanomaterials may be useful to increase in vivo zinc uptake for nutritional applications.Trace elements such as zinc and iron are essential for the proper function of biochemical processes, and their uptake and bioavailability are dependent on their chemical form. Supplementation of trace metals through nanostructured materials is a new field, but its application raises concerns regarding their toxicity. Here, we compared the intracellular zinc uptake of different sources of zinc: zinc sulfate, and ZnO and  Fortiin vitro testing of ENMs, toxicity has been extensively documented [2O3@ZnO nanoparticles in mussel haemocytes, the primary circulating cells of the mussel's blood (haemolymph) that have a similar structure and function to the mammalian macrophage [However, the use of nanomaterials raises concerns about their potential risks. Toxicity assessment of any new application of a chemical must be performed before it is used in commercial products, and this is particularly critical for ENMs, because any modifications of their properties, such as size, surface area, shape and crystallinity, among several others, can significantly impact their toxicity ,10. The cumented –17. As acrophage ,19. Thiscrophage . Using a2.2.1.2O3) coated zinc oxide nanoparticles (nα-Fe2O3@ZnO), with or without inulin coating  were prepared following the procedure reported by Santillán-Urquiza et al. [Saccharomyces cerevisae, FluoZin-3 tetrapotassium salt and Newport Green (NPG) diacetate were purchased from Life Technologies  and stored in aliquots in a freezer at −20°C until their use. Paraformaldehyde was obtained from Electron Microscopy Sciences . All other chemicals were purchased from Sigma-Aldrich  and were used as received.Zinc oxide nanoparticles (nZnO) and haematite α-FeO3 coated2.2.Mytillus galloprovincialis, length 7.1 ± 0.97 cm) were purchased from the Bodega Bay Oyster Company  and were maintained at the University of California Davis Bodega Marine Laboratory  in flow-through seawater tanks for two days before haemolymph extraction. In order to extract the haemocytes non-lethally, a notch was formed in the mussel shell with a triangular file and 0.5 ml of haemolymph was aspirated from the posterior adductor muscle using a sterile syringe containing 0.5 ml of ice-cold physiological saline (PS) Ca/Mg-free . Each aspirated haemolymph was examined with an optical microscope for the presence of haemocytes, determined cell density using a haemocytometer and maintained on ice to avoid clumping. After haemolymph extraction from 3–10 mussels, haemocytes were pooled and density was adjusted to a stock concentration of 5 × 105 cells ml−1 by diluting the cells with PS Ca/Mg-free. Hoechst 33342  was added to stain the cell nucleus. To prepare haemocytes monolayers, 100 µl of haemocytes stock was added into the wells of 96-well plates previously equilibrated at 14°C with 150 µl of PS . Plates were incubated for 45 min at 14°C to allow cell attachment and spreading.Adult mussels (2.3.−1) of different zinc sources: nZnO, nα-Fe2O3@ZnO, with and without inulin coating, ZnSO4  and pure inulin, were prepared in deionized (DI) water and then sonicated in a Branson model 2510 sonic bath  for 30 min (max. 100 W). For the nanoparticle treatments, the concentration was normalized considering only the zinc percentage in each sample, so all the solutions would have the same zinc concentration regardless of their composition. The zinc percentage in each sample was obtained by the results of atomic absorption spectroscopy . The unfiltered stock solutions were then diluted into PS supplemented with 1% fetal bovine serum (FBS) to disperse the chemicals . Six concentrations of each system  were prepared. To analyse the cellular responses due to chemical exposure, 200 µl per well of each target system was added to the adherent haemocytes monolayer (two columns per concentration). Exposures lasted for 120 min at 14°C with gentle mixing.Stock solutions . Lysotracker was added at the same time as the chemical treatment. After 75 min of incubation, JC1, DCF, CAM and EtHD were added; CAM and EtHD were added to the same rows. Then, 25 min later, NPG was added to the rows containing Lysotracker. After incubation for an additional 20 min, the plates were washed three times with 200 µl per well of PS. The fluorescence was detected using a Tecan GENios microplate reader  at the emission and excitation wavelengths indicated in 2.5.n > 50 haemocytes per well) that contained at least one fluorescently labelled yeast inside of the cell (phagocytized).To analyse cell function in the presence of different sources of zinc, haemocyte monolayers (200 000 haemocytes per well) were prepared on a 24-well plate, as described in §2.2. After elimination of non-adherent haemocytes, 1 ml of the corresponding treatment solution was added to a final concentration of 20 ppm along with Alexa-488 yeast (10 yeast/haemocyte). Inulin and PS without nanoparticles were used as controls. Each treatment was replicated in three wells. After incubation at 14°C for 15 min, excess yeast was washed twice with PS and samples were fixed with paraformaldehyde  before imaging using fluorescence (excitation at 488 nm) in a Nikon AZ100 macrozoom stereo fluorescence microscope  at 20× magnification. Images were saved and later visually analysed to determine the percentage of haemocytes  were prepared on a six-well plate, as described in §2.2. After elimination of non-adherent haemocytes, 10 ml of the corresponding treatment solution with a concentration of 20 ppm was added. Inulin and PS without nanoparticles were used as controls. After incubation for 2 h at 14°C, plates were washed four times with PS to eliminate any residual treatment solution. Wells were visually inspected using an inverted microscope throughout the process to determine adherence of haemocytes, with no detectable differences due to either washes or treatment with the corresponding chemical. Tissue was homogenized in 1.9 ml of metal-free filtered seawater pH 1.6  by sonication directly in the well with a probe for at least 2 min, until no intact cells were observed under the microscope. The homogenized tissue was transferred to a 2 ml microcentrifuge tube and samples were incubated for 4 h at room temperature to allow the metal ions to dissolve. Then, samples were centrifuged at 10 000et al. [Zinc concentration in cell extracts was determined by fluorescence using a modified version of the method reported by Grand et al.  which wa2.7.2 per well, 200 000 haemocytes per well). After elimination of non-adherent haemocytes, 800 µl of the corresponding treatment solution with a concentration of 20 ppm was added. The addition of the corresponding fluorescent probe was done following the same conditions described for the plate reader assay (§2.4). After incubation for 2 h at 14°C, slides were washed four times with PS to eliminate any residual treatment solution. Before imaging, haemocytes were fixed with paraformaldehyde . After incubation for 10 min at room temperature, the liquid was eliminated and the slides were separated and compressed under cover glass, then viewed with a 20× water immersion lens on an Olympus BX61WI fixed stage upright microscope using scanning laser confocal microscopy using both interference contrast optics  and the corresponding excitation wavelengths as described previously [For imaging, haemocyte monolayers were prepared in a chamber slide system . Then, the average was divided by the response from the control treatment  to calculate the relative probe response between target and control group.α = 0.05. Analyses were performed using GraphPad Prism 6.0 .Results were statistically evaluated by one-way analysis of variance test. For results from the cellular responses measured in the plate reader, Dunnett's post hoc test was performed for comparisons to control treatments. For internalized zinc concentration and phagocytic activity, Tukey's post hoc test was performed for multiple comparisons. Statistical significance was set at 3.3.1.in vitro has been previously reported for toxicity analysis [There are numerous commercially available fluorescent dyes that can be used to analyse biological cellular responses. EthD-1 is used to determine cell death as it can only enter cells with damaged membranes and its fluorescence increases 40-fold when bound to nucleic acids. Cell viability was determined with CAM, a non-fluorescent membrane permeable dye which upon entering the cell is cleaved by intracellular esterases releasing the non-permeable and strongly fluorescent calcein. JC-1 is used to monitor mitochondrial health as it indicates changes in the membrane potential by changing fluorescence from red to green. Lysotracker Red is a fluorescent acidotropic probe used to detect the lysosomes, which are acidic organelles. H2DCFDA is a fluorescent probe employed to detect reactive oxygen species (ROS), including hydrogen peroxide, hydroxyl radicals and peroxynitrile. Upon oxidation by ROS, the non-fluorescent H2DCFDA is converted to the highly fluorescent DCF. NPG is a cell-permeant probe that selectively binds to Zn(II) and was used to investigate the accumulation of intracellular Zn(II). The use of these fluorescent probes to determine cellular responses analysis –27.Here, we adopted the use of these fluorescent probes to develop a high-content screening (multiple endpoints assessed simultaneously) to determine the impacts of ENMs in mussel haemocytes, cells that can be used as a model system to study the effects of ENMs in marine organisms as well as the cellular toxicity responses ,20. The 3.2.2), gold (Au), titania (TiO2), zirconia (ZrO2), dextran and polyethylene glycol among several others [2O3@ZnO [2O3@ZnO the average size in DI water was 221 nm (70–300 nm) and 85 nm (60–150 nm) for the uncoated and inulin coated version, respectively, while in PS-FBS1%, inulin uncoated and coated nα-Fe2O3@ZnO nanoparticles were 449 nm (102–972 nm) and 324 nm (243–486 nm), respectively. As it was expected, when dispersed in PS-FBS1%, nanoparticle aggregates had a larger size distribution due to increased ionic strength and the presence of proteins, an effect that has been documented in different cell culture media [Coating of a nanoparticle surface is a general strategy used to increase stability, biocompatibility and water solubility as well as to decrease immunogenic response or toxicity. Several inorganic and organic coating materials have been explored to achieve that, including silica  is observed in untreated haemocytes, while treatment with ZnSO4 as a source of soluble zinc caused a very slight increase in fluorescence of NPG in some haemocytes, observed as dim green dots (figure 2b). When haemocytes were treated with nZnO (figure 2c) and α-Fe2O3@ZnO (figure 2e), the increase in green fluorescence was more visible, while treatments with nZnO inulin (figure 2d) and nα-Fe2O3@ZnO inulin (figure 2f), showed the highest fluorescence of all. The results from confocal microscopy strongly correlate with the increase in fluorescence measured with the plate reader.More direct evidence that inulin coated ENM samples increase the amount of intracellular soluble zinc was observed using confocal microscopy of haemocytes exposed to the highest concentration (20 ppm) of the corresponding treatments. As can be seen in figure 22O3@ZnO, in comparison to the uncoated systems or even in haemocytes exposed to the highly soluble zinc sulfate. Inulin coating of nZnO caused a 67-fold increase in zinc accumulation as compared to the uncoated nanoparticle, while haemocytes treated with nα-Fe2O3@ZnO inulin accumulated 15 times more zinc than haemocytes treated with nα-Fe2O3@ZnO. All results suggest that zinc internalization increases were more significant for samples coated with inulin than the uncoated ones, and cannot be attributed solely to the presence of inulin in the media, as there were no significant differences in zinc concentrations in haemocytes treated with nZnO in the presence of soluble inulin in the media. This increase suggests that inulin coating plays an important role in nZnO uptake, either by increasing the dissolution of the nanoparticles, the bioavailability of zinc, or by increasing the active internalization of the nanoparticles by phagocytosis.NPG is a fluorescent probe that selectively binds to intracellular soluble Zn(II), and thus is not able to detect zinc in a nanoparticulate form, as has been previously documented in mammalian cells exposed to zinc oxide nanoparticles . For hae3.4.2O3@ZnO and nα-Fe2O3@ZnO inulin, where 14% and 30% dissolution were obtained, respectively (4 were the ones exposed to the highest concentration of soluble zinc (20 ppm) and accumulated less zinc than haemocytes treated with inulin coated nanoparticles (7.1 ppm of soluble zinc for nZnO inulin and 6.0 ppm of soluble zinc for nα-Fe2O3@ZnO inulin).Zinc oxide nanoparticles have been previously demonstrated to dissolve easily in aqueous media ,34. Howeectively . This inWe hypothesized that active nanoparticle uptake would be the primary cause of increased zinc uptake. The biological role of haemocytes is to defend the mussel from pathogens and parasites, mainly by elimination through phagocytosis . Size plFrom The results obtained by NPG fluorescence as well as the dissolution values in PS indicate that inulin coating has an effect in dissolution, whether in the extracellular media or inside the cells. To further test this, dissolution was analysed in deionized water to determine the direct effect of inulin and eliminate any possible interference of the many components present in the exposure media, PS-FBS 1%. Analysis were conducted at different pHs that mimic different scenarios: neutral conditions present in the extracellular media (pH 7), pH 4.5 that simulates the pH found in lysosomes, and pH 2 that simulates the acidic conditions of human digestive tract.2O3@ZnO. This is in accordance with results obtained in other studies where iron doping of nZnO decreased nanoparticle dissolution and toxicity [2O3@ZnO inulin coating had no effect at pH 4.5 and decreased dissolution at pH 2. Glibowski and Bukowska [2O3@ZnO because its dissolution is lower due to the complexation with haematite. Also, the degradation of inulin itself can affect dissolution by altering the properties of the media by releasing fructose monomers and oligomers, competing with the releasing of free Zn(II) ions from ZnO by formation of stable metal complexes among Zn(II) and the degradation products. It has been previously demonstrated that the presence of organic material in the media affects the dissolution of ENMs, an effect that is also pH dependent [toxicity . Also, aBukowska  reportedependent .Figure As inulin coated nanoparticles are more stable in the media than uncoated ones, they are also more bioavailable, which can lead to increased endocytosis , and can4.2O3@ZnO). Using a novel approach, multiple important cell toxicity parameters  were simultaneously evaluated.A convenient, easy to use and reproducible biological model for the evaluation of biological impact of nanomaterials was used to analyse the cellular responses and toxicity of a set of inulin coated and uncoated inorganic nanoparticles (nZnO and nα-Fe2O3@ZnO may be good candidates for nutritional uses, such as the preparation of fortified foods. Further work aimed to evaluate the uptake of different metal ions using inulin as a coating material is currently being done.Zinc uptake in cells was significantly increased by nanoparticle exposure while having no significant effects on the cellular toxicity endpoints analysed. Inulin coated nanoparticles further increased zinc uptake. These results suggest that inulin coated nZnO and nα-Fe