Nadège Gouignard

Christian Rouvière

Eric Theveneau
email: eric.theveneau@univ-tlse3.fr

Using Xenopus Neural Crest explants to study epithelial-mesenchymal transition

Keywords: neural crest, epithelial-mesenchymal transition, cell migration, adhesion, polarity, dispersion

The epithelial-mesenchymal transition (EMT) converts coherent epithelial structures into single cells. EMT is a dynamic cellular process that is not systematically completed (not all EMTs lead to single cells) and reversible (cells can re-epithelialize). EMT is orchestrated at multiple levels from transcription, to post-translational modifications, to protein turnover. It involves remodeling of polarity and adhesion and enhances migratory capabilities. During physiological events such as embryogenesis or wound healing EMT is used to initiate cell migration, but EMT can also occur in pathological settings. In particular, EMT has been linked to tissues fibrosis and cancer. Neural crest (NC) cells, an embryonic stem cell population whose behavior recapitulates the main steps of carcinoma progression, are a great model to study EMT. In this chapter, we provide a fully detailed protocol to extract NC cells from Xenopus embryos and culture them to study the dynamics of cell-cell adhesion, cell motility and dispersion.

Introduction

In the 60's, during chick embryo gastrulation, epiblast cells were shown to dissociate from the epithelium, lose their epithelial morphology and become mesoderm and endoderm cells [START_REF] Hay | An overview of epithelio-mesenchymal transformation[END_REF][START_REF] Hay | Transformations between epithelium and mesenchyme: normal, pathological, and experimentally induced[END_REF]. The concept of Epithelial-Mesenchymal Transformation was then introduced and defined as a complete loss of epithelial traits, including apico-basal polarity and cell-cell adhesion, accompanied by total acquisition of mesenchymal characteristics (e.g. front-back polarity, cell motility) [START_REF] Hay | An overview of epithelio-mesenchymal transformation[END_REF][START_REF] Hay | Transformations between epithelium and mesenchyme: normal, pathological, and experimentally induced[END_REF]. E-Cadherin, a cell-cell junction protein, and vimentin, an intermediate filament protein, were soon proposed as makers for epithelial and mesenchymal cells respectively. However, a decade later, the term transition was preferred over transformation to reflect the dynamics, the complexity and the plasticity of this mechanism. Indeed, cells were described in various stages of EMT co-expressing markers of epithelial and mesenchymal states or neither.

For a historical perspective on the field of EMT we recommend the review by Angela Nieto in Annual Reviews of Cell and Developmental Biology published in 2011 [START_REF] Nieto | The ins and outs of the epithelial to mesenchymal transition in health and disease[END_REF].

EMT can be initiated by various signaling pathways, including the Bone Morphogenetic Protein (BNP), Wnt, Fibroblast Growth Factor (FGF), Notch and hypoxia pathways, which induce the expression of transcription factors from the Twist, Snail, SoxE, FoxD, Ets and Zeb families. These transcription factors modulate the expression of the proteins involved in cell-cell junctions (type I and type II cadherins) to reduce cell-cell junction strength and promote the loss of apicobasal polarity. They also induce the expression of cell-matrix adhesion proteins (e.g. Integrins), extracellular matrix components (e.g. Fibronectin, Collagen, Vitronectin, Laminin) and remodeling factors (e.g. ADAMs, MMPs) as well as cytoskeleton proteins and regulators (e.g. Keratins, Vimentin, small GTPases) necessary for front-back polarity and motility. In the 90's, a parallel was drawn between tumor progression/metastasis and EMT, identifying common actors and signaling pathways (reviewed in [START_REF] Thiery | Epithelial-mesenchymal transitions in development and disease[END_REF][START_REF] Thiery | Epithelial-mesenchymal transitions in tumour progression[END_REF]). It was suggested that cancer cells were hijacking the embryonic EMT programs. Therefore, a better understanding of the molecular mechanisms regulating EMT was believed to be of primary importance to understand tumor progression.

During the development of vertebrate embryos, EMT is essential for the delamination and long-distance migration of neural crest (NC) cells, a transient multipotent population of cells that arises at the neural plate border and gives rise to a wide variety of derivatives [START_REF] Gouignard | Neural crest delamination and migration: Looking forward to the next 150 years[END_REF]. Over the years, NC cells became a well establish model for physiological EMT, delamination and migration mechanisms, notably for its accessibility and ease of manipulation both in vivo and ex vivo.

In particular, Xenopus NC development recapitulates the main step of EMT and longdistance migration observed in most epithelial cancers. Xenopus NC cells can be defined as the cell population initiating the expressing of a repertoire of EMT transcription factors (Twist1, Snail1/2, Ets1, Zeb2) at the border of the neural plate, at the end of neurulation. Expression of these genes is controlled by the BMP, Wnt, FGF, Notch and hypoxia pathways [START_REF] Steventon | Genetic network during neural crest induction: from cell specification to cell survival[END_REF][START_REF] Steventon | Early neural crest induction requires an initial inhibition of Wnt signals[END_REF][START_REF] Barriga | The hypoxia factor Hif-1alpha controls neural crest chemotaxis and epithelial to mesenchymal transition[END_REF]. Xenopus NC cells then undergo a typical case of cadherin switching, going from E-cadherin-dependent cell-cell junctions to N-cadherin ones [START_REF] Scarpa | Cadherin Switch during EMT in Neural Crest Cells Leads to Contact Inhibition of Locomotion via Repolarization of Forces[END_REF]. This switch is associated with a change of cell polarity from apico-basal, to front-rear, and is directly linked to the acquisition of motility and dispersion, by endowing NC cells with the ability to perform contact-inhibition of locomotion [START_REF] Scarpa | Cadherin Switch during EMT in Neural Crest Cells Leads to Contact Inhibition of Locomotion via Repolarization of Forces[END_REF]. Importantly, EMT in Xenopus NC cells is also linked wtih the acquisition of chemotactic capabilities, since the Hif pathway co-regulates Twist, an E-cadherin repressor, and the expression of CXCR4, the main receptor for CXCL12/Stromal cell-derived factor 1 [START_REF] Barriga | The hypoxia factor Hif-1alpha controls neural crest chemotaxis and epithelial to mesenchymal transition[END_REF][START_REF] Theveneau | Collective chemotaxis requires contact-dependent cell polarity[END_REF]. Finally, Xenopus NC cells express a wide range of matrix remodeling factors including several MMPs and ADAMs [START_REF] Christian | Extracellular metalloproteinases in neural crest development and craniofacial morphogenesis[END_REF].

Altogether, this makes the Xenopus NC cells an extremely relevant model system to study EMT, from its upstream regulators to its downstream effectors and their impact on cell motility.

From a practical point of view, Xenopus produce a high number of large embryos, developing externally, allowing complete access at all times to developmental stages which are hardly accessible in other models. Furthermore, Xenopus embryos allow for gain and loss of function studies, by simple microinjection of Morpholino oligonucleotides, CRISPR/gRNA or mRNA in blastomeres during segmentation. The use of non-toxic fluorescent dyes permits lineage tracing during developmental processes. Using this technique, a cell fate map was generated, allowing reliable targeting of subpopulations of cells, including NC, by injecting specific blastomeres. Finally, NC are easily extracted from developing embryos to be grafted to host embryos for in vivo studes, or cultured ex-vivo in coated petri-dishes in a simple culture medium up to differentiation stages. If dissected at late neurula stage, NC are fully induced and will spontaneously undergo EMT-driven dispersion and migration during the first few hours following grafts or culture. EMT and migration of NC cells can be monitored in vivo or ex vivo using time-lapse cinematography.

In this book chapter, we describe the protocol to extract NC cells for ex vivo culture and discuss how to monitor and analyze NC dispersion and migration from time-lapse images.

Materials

Ex-vivo culture of Xenopus Neural Crest cells

Extracellular matrix preparation

1. Petri dishes (any dish from 35 mm to 90 mm made of untreated plastic) or multi-well dishes (e.g. LabTek, Ibidi µSlides 80821 or equivalent).

2. Purified Fibronectin in solution (Sigma F1141 or equivalent reference from other providers).

3. Phosphate Buffer Saline (PBS) 1X: Start with 800 ml of distilled water add 8 g of NaCl, 0.2 g of KCl, 1.44 g of Na2HPO4, 0.24 g of KH2PO4, adjust the pH to 7.4 with HCl, add distilled water to a final volume of 1 liter. 5. P20 micropipette. 6. A small uncoated petri dish filled with culture medium to collect NC explants.

Amphibian media and culture media

1. Normal Amphibian Medium (NAM) stock solutions, must be autoclaved and can be stored at room temperature. In distilled water, NAM A (NaCl, 1.1M; KCl, 20mM; NaHCO 3 , 10mM), NAM B (MgSO4, 10mM; CaCl2, 10mM), NAM C (EDTA, 1mM), NAM D (Na 2 HPO 4 , 16mM; NaH 2 PO 4 , 4mM).

2. Working solutions: NAM 0.1X (for one liter add 10 mL of each NAM stock solutions and add 960 mL of distilled water), NAM 0.25X (for one liter add 25 mL of each NAM stock solutions and add 900 mL of distilled water).

3. Culture medium -Danilchik's for Amy (DFA): 53 mM NaCl, 5 mM Na 2 CO 3 , 4.5 mM KGluconate, 32 mM NaGluconate, 1 mM MgSO4 (7H 2 O), 1 mM CaCl2, 0.1% BSA; adjust pH to 8.3 with 1 M Bicine. Store at -20°C. After thawing, add 1000 U penicillin and 100 μg/ml streptomycin.

Time-lapse imaging

1. Microscopes: inverted, upright microscope, dissecting microscope (see Note 1).

2. Camera and software.

3. Temperature controlled room (see Note 2). 4. Formaldehyde.

Analysis of cell migration and dispersion dynamics

1. A computer equipped with a modern CPU and with sufficient amount of RAM and storage space to process the data. The total RAM available should be twice the size of your largest dataset to allow opening and processing of the data.

2. A free (FIJI/Image J) or commercial software (e.g. Imaris/Bitplane) for automated detection of nuclei, tracking and dispersion analysis.

Methods

Ex-vivo culture of Xenopus Neural Crest cells

Extracellular matrix preparation

1. Aliquot the Fibronectin solution as a 10X stock by 20 or 50 µL in 500 µL centrifuge tubes and store at -20°C. For coating on glass coverslips, the 10X stock is at 1 mg/mL. For coating on plastic petri dishes, the 10X stock is at 100 µg/mL. When making the 10X stock for plastic, use PBS1X containing 0.1% BSA for long-term stability of the solution.

2. On the day of the experiment, select a dish appropriate to your microscope (see Note 3).

3. For glass dishes/coverslips dilute your Fibronectin in PBS1X to a final concentration of 100 µg/mL, for plastic, to 10 µg/mL. Directly thaw the frozen aliquot of Fibronectin by adding the required amount of PBS1X at room temperature in the Fibronectin aliquot. (e.g. add 450µL of PBS1X on top of a 50µL aliquot of 10X Fibronectin to obtain 500µL of Fibronectin 1X for coating) (see Note 4).

4. Place the dish on a heating block for 1 hour at 37°C.

5.

Remove the fibronectin solution using a P1000 micropipette and rinse once with PBS1X (see Note 5).

6. Rinse the dish with a small amount of DFA1X to remove traces of PBS1X.

7. Fill the dish with DFA1X.

Microdissection

1. Place embryos at stage 15/16 in a petri dish filled with NAM 0.25X and remove the vitelline membrane using fine forceps. Let them recover as superficial wounding may have occurred and let embryos further develop to stage 18 (see Note 6, Movie 1).

Select embryos at stage 18 (Movie 2).

3. Fill the dish containing the modelling clay with NAM 0.25X. Using the glass ball, form small holes in the modelling clay. The depth of each hole should be roughly half the width of an embryo (see Note 7, Movie 3). 4. Place one embryo per hole. Turn each embryo slightly on one side so that the neural crest region to be removed is facing up (Fig. 2a, Movie 3).

5. Using the glass ball, move gently the clay towards the embryo until it touches the epidermis making sure the orientation is preserved (Fig. 2b) then apply pressure all around the embryo by pushing the clay onto its sides to firmly secure the embryo (Fig. 2c, Movie 3, see Note 8).

6. Using the hair knife make a small incision posteriorly and dorsally to the neural crest area (Fig. 2c 9. Place the hair knife parallel to the ventral side of the neural crest region (dotted line on Fig. 2d). Apply a gentle pressure (do not attempt to cut the tissue) and move from lateral to medial repeatedly. The neural crest region will detach from its surroundings (Fig. 2e-e'). Continue until the whole region detaches by itself (Movie 2). After dissection there should be a hole in which the underlying mesoderm can be seen (Figure 2f). When a dissected embryo is released from the clay the actual size of the wound should be relatively small and the neural tube on the dissected side should be intact (Figure 2g-g'). Look out for contamination from other cell types (Fig. 3, see Note 9). An example of a rough dissection including mesoderm is shown at the end of Movie 3. 10. Using a P20 micropipette set to 10 µL rapidly transfer the explant into a dish containing DFA1X (Movie 3).

11. Repeat steps 3 to 10 to collect multiple explants.

12. Cut the explants in small pieces using the hair knife and fine forceps (Movie 4). A complete unilateral neural crest region can be cut into 4 to 5 pieces each containing ~100 -200 cells.

13. Transfer the explants onto the Fibronectin-coated dish.

14. Array the explants to facilitate monitoring of the explants under the microscope (see Note 10).

15. Let the explants adhere to the substrate for at least 15 minutes before moving the dish.

Time-lapse imaging

1. Gently place your dish onto the microscope.

Analysis of cell migration and dispersion dynamics

Analysis of cell migration

1. A nuclear tracer (e.g. H2B-mCherry/GFP) must be injected into early Xenopus embryos prior to dissection if single cell tracking is to be performed (see Note 13). Manual or automated tracking can then be done using ImageJ/FIJI (Manual tracking or trackmate plug-in) or commercial alternatives such as Imaris/Bitplane (spot tracking algorithm).

2. From tracks, cell speed and directionality can be retrieved. This information can then be correlated with the dynamics of cell dispersion (see Note 14).

Analysis of cell dispersion using

the total explored area over time (Fig. 4) 1. In ImageJ/FIJI use the "image\stacks\tools\make substack" tool to extract images from each movie at regular interval (e.g. first image and then one image per hour). Save all substacks (one per XY position) into a separate folder for analysis (Fig. 4a).

2. In ImageJ/FIJI, link all external cells of an explant using the broken line tool and measure the area. Repeat this step for each time step of the substack (Fig. 4b-c). Repeat for each substack movie. Save the results as .xls or tabulated text file.

3. If only one experimental condition is analyzed over time, plotting the data as box whiskers plots for each time point will allow variability at each time point and overall dispersion over time to be assessed. If multiple conditions are to be plotted in parallel on the same graph, curves representing the mean area per time point alongside the standard deviation are more practical.

Analysis of cell dispersion using triangulation between nearest neighbors

1. In ImageJ/FIJI use the "image\stacks\tools\make substack" tool to extract images from each movie at regular interval (e.g. first image and then one image per hour). Save all substacks (one per XY position) into a separate folder for analysis.

2. In imageJ/FIJI, go to "analyze/set measurements" and make sure "centroid" and "area" are selected.

3. Open one of the substacks and estimate the mean area occupied by one nucleus in your samples by measuring about 20 different nuclei using the elliptical or polygon selection tools in ImageJ/FIJI.

4. Go to "analyze/analyze particles". Set the minimum and maximum area occupied by a nucleus as measured in step 3 and press OK. This will produce a list of XY coordinates of all nuclei detected. 5. Use the Delaunay/Voronoi plug-in in ImageJ/FIJI to build triangles from coordinates produced by the "analyze particles" tool. The options "infer selection from particles" and "make Delaunay ROI" must be selected.

6. Go to "analyze/tools/ROI manager" and do the following steps: "Add", "Split", "multimeasures".

7. Save the results table containing the areas of all triangles. It is important to note that the first value of that list is the cumulated area occupied by all triangles (see Note 15.) 8. If this has to be done on multiple images, a macro can be used. A macro for batch processing and a custom-made Look-up table (LUT) for color-coding of triangles were developed in the Theveneau lab and are available upon request.

The respective merits of the two methods to analyze dispersion are discussed in Note 16.

Notes

1. In Xenopus NC cells the dynamics of dispersion depends on the timing of cell-cell dissociation, which is linked to the E-to-N-cadherin switch. Thus, the timing (how long it takes) and intensity (are all cells becoming individual?) of dispersion are a proxy for EMT dynamics.

This can be assessed on low magnification images even without time-lapse if several conditions are plated at the same t0 and compared at a similar time point after the beginning of the cell culture. Therefore, monitoring simultaneously several explants at low magnification on a dissecting microscope could be sufficient for a first screening of experimental conditions. However, if dispersion analysis is to be performed using single cell detection by nuclear staining or if cell tracking is to be done in parallel, then higher magnifications (in XY and time) are required and a motorized stage is necessary. A wide field microscope with a 10X objective will allow visualization of NC explants the size of a fifth of a complete unilateral NC region, while providing enough XY resolution to track single cells accurately using a time interval of 3 min. If cells are labelled with a membrane tracer and the dynamics of cell-cell contacts is to be analyzed, 20X or 40X objectives are needed and shorter time intervals are necessary (1min or below). Inverted microscopes are the usual choice for cell culture assays. However, we obtained very good results using upright microscopes with dry lenses. In this case, cells need to be cultured in a dish that can be filled entirely with culture medium, without any air bubbles, and flipped upside down. Several providers have culture dishes with tight lids in their catalogue. If your cell culture device of choice does not exist in a version with a tight-fit lid, it is possible to seal the lid using high vacuum silicone grease. This type of upside-down cell culture removes all dead and/or non-adherent cells, keeping the substrate clean for the healthy cells. However, this set-up is not appropriate if experimental conditions impairs cell-matrix adhesion as cells would detach and thus be lost.

2. Xenopus embryos can develop between 12 and 28°C, the optimal range being 14 to 25°C. NC explants are a bit less flexible and usually survive and migrate better between 18 and 21°C.

Below 18°C cell-cell dispersion is affected and explants usually remain as pseudoepithelial sheets for much longer than they would in vivo. Above 21°C, cell survival is lower. Between 18-21°C, explants roughly the size of a fifth of one unilateral NC region dissected at stage 18, spread extensively during the first two hours and then disperse as small clusters and eventually as single cells.

3. On upright microscopes, use tight-fit lid petri dishes that can be completely filled with culture medium and closed hermetically or open dishes with a water-immersion lens. For inverted microscopes, any type of multi-well dishes can be used depending on the type of motorized stage. In our hands, the 8-well Ibidi µSlides (80821) gave the best results. They allow culture in small volumes (300µL) suitable for drug screening. They are made of a polymer that allows low concentration of Fibronectin coating for plastic to be used while being compatible with confocal imaging.

4. When coating large dishes do not cover the entire dish with Fibronectin. Use a marker pen to delimitate an area where Fibronectin will be coated. A circle with a diameter of 1 cm is enough to plate 5 rows of 5 explants and can be coated with a 500µL drop of Fibronectin 1X solution.

Always cover the dish during Fibronectin incubation at 37°C to avoid evaporation. If very large dishes are used (e.g. 90 mm bacteria plates), place a piece of wet paper inside the dish to keep it moist.

A successful Fibronectin coating can be assessed by eye. When the Fibronectin solution is

removed at the end of the 1-hour incubation, the part of the dish or the well that is coated remains slightly wet and appears shiny when exposed to light whereas a similar incubation with a drop of water or PBS1X will leave the dish dry as soon as the liquid is removed. The area that received water or PBS cannot be distinguished from the rest of the dish.

6. Dissection of Xenopus NC explants is very easy when the embryos are at the right developmental stage. Just prior to migration, NC cells are not significantly attached to their surrounding tissues (ectoderm, mesoderm, ectodermal placodes). Yet, they strongly attach to each other. This allows the whole NC region to be removed, as it is an epithelial structure only loosely connected to the other tissues. The best stage is 18. At this stage the neural folds nearly finished closing (Movie 2). Only the most anterior part of the neural plate is still open and has the shape of a tear drop. The NC region appears as a bump lateral to the anterior neural plate and extends roughly to the middle of the anteroposterior axis. Before that stage, the NC region is still attached to the neural plate and one would need to arbitrarily cut the explant out of the embryo without any clear landmark to use as reference. After stage 18, cells initiate migration and start adhering strongly to the mesoderm and the ectoderm. First, many cells are lost while peeling the superficial ectoderm. Second, it increases the probability of mesoderm contamination (Fig. 3, Fig. 5, last part of Movie 3). Dissections at early (prior to stage 18) or late stages (after stage 18) are still technically possible, but they require an extensive amount of training. If such stages are absolutely needed for a given project, one should train dissecting by placing embryos at similar stages that have been stained with a neural crest marker (e.g. slug, twist) next to the embryos to be dissected, for reference. 7. Embryos should not be put completely inside the clay. It is important that roughly a third of the embryo still pops out of the modelling clay after the embryo has been immobilized. Otherwise, the region of interest will be difficult to access for dissection with the hair knife.

8. Once the embryo has been immobilized by applying pressure with the modelling clay, all previous morphological landmarks are gone. Thus, it is critical that the orientation of the embryo to be dissected is correct before applying pressure. 9. At the end of neurulation (stage 18), NC cells adhere to each other more than they adhere to their surrounding tissues (see Note 6) making dissection relatively easy. Yet, contamination from adjacent cell types can sometimes occur. Mesoderm and endoderm are richer in vitellus and appear whiter than NC cells, which are comparatively translucent and grey. The last part of Movie 3 shows an example of a rough dissection during which a bit of mesoderm is taken out together with the neural crest. See also Fig. 3 and its legend to see how to proceed when mesoderm is mistakenly dissected alongside NC cells. Superficial ectoderm may be detected by the presence of dark spots corresponding to the pigments. Explants should always be screened for contamination at the end of the culture and explants with contamination from other cell types should be excluded. In 2D-culture, ectoderm, neural plate, neural crest, mesoderm and endoderm can be easily recognized morphologically using several criteria such as the shape of the cells and nuclei or the size of vitellus platelets (Fig. 5). Non-neural ectodermal cells flatten extensively onto the substrate and form a continuous monolayers with large nuclei. Neural plate explants remain as a tight cluster and project axons within few hours of cell culture. Mesodermal and endodermal cells are significantly larger and whiter than neural crest cells. They contain large vitelline platelets. 10. When plating explants next to each other onto Fibronectin, do not put explants to close to one another as NC cells can attract each other via a process called co-attraction or mutual attraction. This would lead neighboring explants to merge. A rule of thumb is to make sure that each explant is at least at 5 times its own diameter away from the nearest explants in the dish.

11. If only overall explant dispersion is to be analyzed, time-lapse imaging is not necessary.

Explants can be fixed after several hours and the dispersion can be analyzed using triangulation, total explant area or nearest neighbors analysis. However, having the dynamics of the population and the movement of single cells can be very informative. For movies done with a 10X objective at a temperature between 18-21°C, cells migrate at around 1-3µm/min and a time interval of 3 minutes is optimal for cell tracking (either manual or automated). 8-hour movies at 21°C are enough to see the main steps of dispersion: adhesion, spreading, break-up of the explants as clusters and single cell dispersion. If under control conditions single cell dispersion does not occur within this time frame it can be due to several possibilities: the temperature is too low, cells were extracted at earlier stages than 18 and have not yet completed their cadherin switch (there is a sharp decrease of the amount of E-cadherin from stage 15), the substrate is not optimal for migration and cells have problems moving away from each other. 12. To fix cells at the end of a culture experiments do not remove the DFA1X, instead add directly formaldehyde into the dish. In 8-well slides, if each well contains 300µL of DFA1X, simply add 100µL of formaldehyde 16% in each well. In larger dishes, add locally formaldehyde 16% atop the explants to pre-fix the cells, wait 5 minutes, remove the medium, fill with formaldehyde 4% and further fix for 30 minutes. 13. To label NC cells prior to cell culture, one can inject at either 2, 4, 8, 16 or 32-cell stage. If simple tracers to label nuclei and/or membranes are used, the easiest way is to inject two blastomeres at 2-cell stage and select the brightest embryos at stage 16 prior to removing the vitelline membrane. If nuclear/membrane tracers are to be used in combination with other treatments that may affect early development, it is recommended to inject at 8-cell stage or later.

From 8-cell stage the animal blastomeres only contribute to the ectoderm (neural plate, NC, placodes, epidermis), therefore injections at 8-cell stage or later will leave the mesoderm and endoderm intact, reducing possible side effects on gastrulation.

14. In control conditions, cell-cell dissociation leads to a progressive shift from collective/pseudoepithelial-like behavior, to a collective/mesenchymal behavior, to single cell behavior. Individual cell directionality decreases in single cells compared to cells within a group, whereas individual cell speed increases. This, in control conditions, typically correlates with the rate of expansion of the explant. The initial slow-paced directional radial expansion turns into a fast disorganized dispersion of single cells. Overall dispersion comes to a halt when local cell density is such that cells have an equal probability of moving in any direction. However, under experimental conditions, one can interfere with cell-cell or cell-matrix adhesion and affect the relationship between explant dispersion and single cell behavior. For instance, reducing cell-cell adhesion may promote single cell behavior while overall radial expansion of the explant is impaired. Thus analyzing single cell behavior and explant dispersion in parallel can be very informative.

15. Triangles' areas follow an exponential distribution, thus the usual statistical tests for normal/Gaussian datasets do not apply if one wants to compare the distribution of triangles' areas from one explant to another. This problem is easily solved when comparing populations of explants. One should plot the mean triangle area of each explant per experimental condition.

That way each explant has the same weight in the mean of a given condition (otherwise explants with more cells contribute more triangles and can skew the mean of the dataset). In addition, the distribution of mean areas under control condition follows a Gaussian distribution and allows simple statistical tests to be performed. 16. Analyzing explant dispersion using the total explored area is the simplest and most effective way. This technique does not require pre-or post-labelling of cell's nuclei. It enables the inclusion of the impact of all cells, and can be done on simple low resolution bright field or phase contrast images. Triangulation on the other hand has several caveats. First, one needs to detect single cell nuclei. This can be done by labelling NC cells with a nuclear tracer or post-staining them with DAPI. Automatic detection of nuclei is possible (e.g. analyze particles plug-in in Image J/FIJI) but weaker nuclei are often not detected, artificially increasing the area of some triangles.

Similarly, areas with high cell density cannot be properly segmented and this results in artefacts (Fig. 4). This later problem can be partially solved by acquiring images on a confocal microscope, although high resolution images of nuclei often confuses the automated detection of particles, since pixel intensity within each nucleus can vary substantially, and nuclei no longer appear as solid structures. Alternatively, one can mark each cell's nucleus manually. The first option, using confocal imaging, will never be a realistic routine procedure, even if one owns a dedicated confocal microscope for time-lapse imaging. The second option, manual marking of individual nuclei, is extremely time consuming, especially if one wants to perform triangulation at multiple time points for multiple explants to assess the dynamics of dispersion. A typical explant has between 100 and 200 cells. If one wants to perform triangulation on 20 explants at 5 time points each, it means marking manually between 10000-20000 nuclei. This increases exponentially if one needs to do experiments in triplicate and has to compare multiple experimental conditions. Thus, if one absolutely needs to use triangulation (e.g. local differences in terms of distance between neighbors within each explant are important), we recommend using the automated detection of nuclei. However, it is important to bear in mind that some nuclei will not be detected regardless of the technique used to mark them, or to detect them. It is important to first establish the conditions for automated detection on a small subset of data and to compare the overall output with the one obtained when marking nuclei manually. A difference of more than 5% should not be considered acceptable and image acquisition and nuclei detection would need to be improved before embarking on large scale automated detection of nuclei. Step 1, make a superficial incision using the tip of the hair knife. Step 2, insert the hair knife underneath the skin, parallel to the embryo and move anteriorly. Do not stab the embryo towards the inside. Step 3, gently move from medial to lateral to remove the pigmented layer. Avoid using the tip of the hair knife, rather work with the side of the hair. d, The embryo after the superficial pigmented layer has been removed. The green dotted line indicates where the hair knife should be positioned to remove the neural crest. Slightly press down using the side of the hair knife and move from lateral to medial (step 4, green arrows). e, The NC explant (nc) has been lifted, the neural tube (nt) and mesoderm (m) are visible, see the zoom-in in e'. f, the NC explant has been removed, seen in f', the dissected region is shown in f''. g-g', the embryo after dissection and freed from the clay. Scale bars: a-g, 1 mm; e', f'' and g', 150 µm; f', 500 µm. The endoderm (end) lining the internal cavity is visible. b', Zoom of the mesoderm contaminated NC explant. The dotted line delineates the mesoderm which appears whiter than the neural crest. b'', Zoom of the endoderm at the site of the dissection. c, most of the mesoderm can be removed using the hair knife prior to detaching the neural crest explant but some mesoderm may remain (m, arrow). d, NC explant after being detached from the embryo, remaining mesoderm contamination is indicated by the arrows. e-g, Trying to clean the small bits of mesoderm is too difficult. Instead, cut the neural crest explant in two pieces and discard the half containing the mesoderm contamination. The explant on the right-hand side of panel g is good for culture, the one on the left-hand side should be discarded.

References Figures Legends

Movies

4 .

 4 Bovine Serum Albumin (BSA). 5. Heating block. 2.1.2 Microdissection (Fig. 1) 1. Hair knife. Dip the tip of a short glass Pasteur pipette into melted wax and inset the root of a hair (eyebrow, eyelash or else) in the tip of the pipette before the wax has set. 2. A dish filled with modelling clay. 3. Glass ball. Prepared by melting the tip of a short glass Pasteur pipette. 4. Fine forceps.

  step 1, Movie 3). 7. Insert the tip of the hair knife in that initial hole, keeping the hair parallel to the tissue. The hair knife should be in between the pigmented layer and the neural tissue (Fig. 2c step 2, Movie 3) 8. Gently remove the pigmented layer from medial to lateral to uncover the neural crest region (Figure 2c step 3, Movie 3).

2 .

 2 Check explants viability and fluorescence if embryos were microinjected with a tracer prior to dissection. 3. Adjust lighting conditions (light intensity, exposure time). 4. Record the XY positions of each explants. 5. Set the time interval and total duration of the movie (see Note 11). 6. Start the acquisition. 7. At the end of the movie, cells can be fixed in formaldehyde 4% for further analysis and staining (see Note 12). Typical dispersion of control neural crest explants is shown in Movie 4 and Fig. 4.

Figure 1 -

 1 Figure 1 -Tools for microdissection of Neural Crest explants

Figure 2 -

 2 Figure 2 -Microdissection of neural crest cells: an ideal case.

Figure 3 -

 3 Figure 3 -Microdissection of neural crest cells: how to handle mesoderm contamination.

Figure 4 -

 4 Figure 4 -Analyzing the dynamics of cell dispersion

Figure 5 -

 5 Figure 5 -How to distinguish neural tube, skin, placodes and mesoderm from neural

Acknowledgements

Eric Theveneau and Christian Rouviere are permanent CNRS staff. Work in the Theveneau lab is supported by the Region Midi-Pyrenees (Installation Grants for Excellent Researchers, 13053025), the Fondation pour la Recherche Medicale (AJE201224), the CNRS and the Université Paul Sabatier. Nadège Gouignard is the recipient of an individual fellowship from FRM (ARF20150934153) and the Marie Curie Prestiges Program (PRESTIGES 2015-4-007).


Mohamad El-Roz

Louwanda Lakiss

Jaafar El Fallah

Oleg I Lebedev

Frederic Thibault-Starzyk

Valentin Valtchev

Incorporation of clusters of titanium oxide in Beta zeolite structure by a new cold TiCl4-plasma process: physicochemical properties and photocatalytic activity

Keywords: [year], [vol], 00-00

Introduction

Over the last few years photocatalytic processes using titanium dioxide (TiO2) as photocatalyst have attracted special interest due to their numerous applications in water and air purification. [1][2][3][4][5][6] Because of its high activity and stability, low cost and non toxic nature, titanium dioxide is considered as a very promising photocatalyst. When exposed to light in the presence of oxygen, it produces highly oxidative radical species that successfully oxidize most of the organic pollutants present in air and water into inert ultimate products such as CO2 and H2O. Despite these valuable properties, the use of TiO2 for industrial purposes remains limited due to the photocharge recombination [7] and adsorption/desorption processes of the reactants and reaction products [8]. It has also been reported that particle size distribution and dispersion of titanium dioxide particles have a direct impact on the efficiency of the catalyst. In this context, researches focused mostly on the deposition of titanium dioxide onto different types of support not only to respond to the needs of highly dispersed TiO2 nanostructures but also to exploit the adsorption properties of supports that might improve the photocatalytic activity. Several groups reported the preparation of TiO2 supported onto different types of adsorbents such as fiber glass [9][10], natural fiber [11], activated charcoal [12][13], alumina, silica or zeolite materials. [14][15][16][17][18][19][20] Among these supports, zeolites were found to be the ideal candidate due to their high specific surface area and micropore channels that provide confined space for stereoselective reactions. In addition, the physicochemical properties of zeolitic materials can be easily modified either by in situ or by post-synthesis methods. They are also eco-friendly and their tunable hydrophobic/hydrophilic character provides selective exclusion of undesired molecules or ions [21,22]. Recent studies have demonstrated the promising 50 photocatalytic efficiency of TiO2/zeolite composites [17,[23][24][25] and interesting photovoltaic applications of clusters of titanium dioxide encapsulated within zeolites [26]. TiO2/zeolite supported photocatalysts can be prepared by different methods that are classified into three main groups: (i) 55 solid state dispersion methods (SSD) or mechanical mixing; [17,[27][28][29] (ii) post-synthetic approaches as dealumination, followed by a vapor-thermal treatment using a TiO2 precursor [30]; and (iii) in situ synthesis approaches where TiO2 nanoparticles are anchored at the zeolite surface during the hydrothermal treatment 60 [14][15][16]. Plasma treatment (thermal and non thermal) has also been used to prepare nanosized TiO2 supported thin films (silica wafer, glass wafer…) [31][32][33][34].The most often employed TiO2 precursor, for this treatment, is the titanium tetraisopropylat that is delivered in 65 gas phase comprising O2, O2/Ar, O2/H2O [31][32][33][34]. As known, during the plasma ionization of an oxygen-containing gas phase, oxidative species (O2 •-, O, O3…) are generated. These species are able to oxidize the titanium precursor into crystalline TiO2 that is deposited on the surface of the support. The formation of 70 relatively large TiO2 particles limits the preparation of efficient TiO2/zeolite composites because of the difficulty to incorporate TiO2 in zeolite pore system. The present work reports the use of a modified cold TiCl4-plasma approach that allows an efficient incorporation of clusters of titanium dioxide in the pore system and on the surface of zeolitetype materials. In contrast to the traditional plasma treatment described above, the present approach consists in the incorporation of atomic scale titanium species in zeolite channels.

It is based on a direct-decomposition of the titanium vapor precursor (TiCl4), by plasma (TiCl4-plasma) in absence of any other carrier gas, at low pressure (<0.5 mbar) and at relatively low temperature (<350 K). After the TiCl4-plasma process, an O2-plasma treatment is performed in order to oxidize the already 10 deposited titanium species that are anchored to zeolite structure favoring then the TiOx formation by generating high oxidant species [35]. The elaborated TiOx supported catalysts (Ti-Beta) were subjected to detailed characterization prior to be tested as photocatalysts for methanol photooxidation. For comparison, a reference sample prepared by SSD method (TiO2/Beta) was employed.

Experimental part 20

Synthesis and post synthesis treatment

Zeolite Beta synthesis: Nanosized Beta crystals (Beta) were synthesized from a colloidal precursor suspension having the following chemical composition: 0.35 Na2O: 4.5 (TEA)2O: 1 Al2O3: 30 SiO2 : 295H2O. The silica source for the preparation of the initial precursor suspension was freshly freeze-dried colloidal silica Ludox SM 30 (30 wt %). Aluminium isopropoxide (98%, Aldrich) was used as an alumina source and tetraethylammonium hydroxide (TEAOH, 20 wt% in water, Merck) as a structuredirecting agent. These components were mixed under vigorous stirring for 15 min and aged on an orbital shaker at ambient temperature for 24 h prior to the hydrothermal treatment performed at 373 K for 8 days. The nanosized crystals resulting from the hydrothermal treatment of the colloidal suspension were purified by three steps centrifugation (20,000 rpm, 60 min), decanting of the supernatant and redispersion in distilled water using an ultrasonic bath. The resulting suspension was freezedried to recover the sample in a powder form. The Beta zeolite crystals were then calcined to remove the organic template. The calcination process consisted in heating the sample from RT to 40 823 K with a heating rate of 1.75 K/min, kept at 823 K for 5 hours and then cooled down to room temperature in 5 hours.

Preparation of Ti-Beta:

The incorporation and encapsulation of titanium oxide in calcined Beta zeolite was performed using a plasma process in a Pyrex reactor (dielectric barrier discharge plasma with a 50 Hz sinusoidal power supply (2 kV)) (Scheme 1-A). After introducing the powder in the reactor, the system was evacuated until the pressure stabilizes at ~10 -2 mbar. Then TiCl4 was introduced under continuous vacuum in order to reach an equilibrium pressure close to 0.5 mbar in order to purge the plasma reactor. The cold plasma was then turned on to decompose the TiCl4 precursor in absence of any other gas. It was maintained about 5 to 10 seconds and then the valve of TiCl4 tube was closed in order to remove the excess of TiCl4 and the 55 different species formed during plasma. The process was repeated three times. It should be noted that in our conditions the plasma was shut-off when the partial pressure of TiCl4 exceeded 0.7 mbar. The TiCl4plasma treatment was followed by O2-plasma treatment for more 60 than 10 minutes (depending of the sample amount) at low pressure (3.5 mbar). During the experiments, the temperature increase due to plasma process up to 393 K. The obtained material was named "Ti-Beta". For comparison three types of samples were also investigated as 65 references: a TiO2 sample (P25 from Degussa), an initial zeolite Beta sample (used for the preparation of Ti-Beta), and a TiO2/Beta (15/85 wt. %) prepared by Solid State Dispersion method using ethanol as solvent.

70

Characterization

In order to investigate the crystallinity and phase purity of the samples, calcined and treated Beta-type crystals were subjected to X-ray diffraction (XRD) analysis using a PAN analytical (X-Pert Pro) diffractometer with CuKa1 source (l=0.15406 nm, 40 kV, 75 30 mA). The morphological features and the chemical analyses of the samples were studied using Scanning Electron Microscope (SEM, Hitachi S3460) equipped with an energy dispersive spectroscopy (EDS, ThermoNoran) device. Beta and Ti-Beta materials were also characterized by transmission electron 80 microscopy (TEM), including electron diffraction (ED), high resolution TEM (HRTEM) and energy dispersive x-ray (EDX) analysis, using a Technai G2 30UT microscope equipped with an EDAX X-ray microanalysis unit, operating at 300 kV and having 0.17 nm point resolution. The TEM samples were prepared by 85 dispersing the powder onto a copper grid coated with a holey carbon film using ethanol as solvent. Low intensity beam conditions were applied for TEM measurements in order to minimize the degradation and amorphisation of the material under the electronic beam.

90

Nitrogen sorption measurements were carried out at 77K on a Micromeritics ASAP 2020after degassing at 423 K for 24 h. The surface areas were calculated using BET equation and the total pore volumes of the samples were estimated at a relative pressure of 0.99. The samples were also investigated by solid state NMR 95 spectroscopy. MAS 29 Si and CP MAS 1 Hà 29 Si NMR experiments were conducted at 300K on a BRUKER ADVANCE 400 DSX spectrometer operating at 400 MHz-9.4 T for the 1 H Larmor frequency, and equipped with a BRUKER 4 mm 1 H/X solid-state CP MAS probe. A representative sample (~ 50 mg) 100 was placed in a 4-mm zirconium rotor. The Magic-angle spun at 14 kHz with pulse duration of 3.5 ms, contact time of 6 ms and relaxation time of 1s. The number of scans was adjusted to ensure a signal-to-noise ratio of at least 1024. For MAS 27 Al NMR experiments, the spectra were recorded with a spinning speed of 105 about 12 kHz. Chemical shifts were referenced to a 0.1 M Al(NO3)3 aqueous solution. In order to investigate the surface modification of Beta-type zeolite during plasma treatment, in situ FTIR spectroscopy was performed on self-supporting wafers (20 mg/2 cm 2 ) placed in an 110 in situ FTIR-plasma reactor (Scheme1-B). First, the pellet was subjected to evacuation under vacuum in order to evacuate the atmospheric gas present in the ionization chamber and to remove water weakly adsorbed on Beta surface. The first dose of TiCl4 was introduced by opening the valve for ~10 s to reach a pressure of ~0.50 mbar under evacuation. This step allowed the purge plasma reactor from the residual gases that could remained. The IR/plasma experiment was performed in the same order described above (see "post synthesis treatment" part) but for a shorter time due to the low amount of sample (pellet mass ~20 mg). The IR spectra were collected in transmission mode on a Bruker Vertex 80v equipped with a cryogenic MCT detector (4 cm -1 resolution, 8 scans, and acquisition time ~1.2 s). The acidic properties of the resulting materials were investigated by IR using pyridine as probe molecule. Powders were pressed (~10 7 Pa) into self-supported discs (2 cm 2 area, ~20 mg) and placed in an IR cell equipped with KBr windows. IR spectra were recorded using a Nicolet 6700 IR spectrometer equipped with a MCT detector and an extended-KBr beam splitter was used. Spectra were recorded in the 400-5500 cm -1 range at a resolution of 4 cm -1 and 128 scans were collected for each spectrum. A movable quartz sample holder allowed placing the self-supported discs in the infrared beam, for recording spectra, and moving it into a furnace at the top of the cell for thermal treatment. Pyridine (Py) adsorption was performed by introduction of doses inside the infrared cell containing the previously activated (under vacuum at 473 K for 5 h) self-supported discs of the samples. After introduction of each dose of Py, the Beta sample was heated at 473 K for 10 min to allow diffusion toward all accessible sites before recording spectrum. Infrared spectra were recorded after Py saturation (1.33 mbar at equilibrium) followed by evacuation at 473 K to remove physisorbed species. In all experiments, analytical grade pyridine (Aldrich) was used after water trapping with molecular sieve 3A. The decomposition of the massif into overlapping IR bands of adsorbed pyridine on acidic sites and the calculation of the IR band area have been performed using Omnic v8.2 software. The values were normalized for the same weight (20 mg) of pellets. The accessible silanol sites were exchanged with heavy water vapor (D2O) (10 mbar at equilibrium followed by an evacuation 40 under vacuum at RT). This process was repeated 3 times in order to obtain a total H-D exchange of all accessible silanol sites. After exchange and evacuation, IR measurements were performed on a Nicolet 6700 IR spectrometer equipped with a MCT. Spectra were recorded at 4 cm -1 and 128 scans were co-45 added for each spectrum.

Photocatalytic tests

In order to test the photocatalytic activity of Ti-Beta materials, photocatalytic oxidation of methanol was performed. Ti-Beta 50 catalyst was pressed into self-supported wafers (2 cm 2 , m~20 mg) and put in the photocatalysis reactor. It was made of a stainless steel cylinder that carries a toroidal sample holder in its centre, where the catalyst self supporting wafer was placed. Tightness was obtained by Kalrez© O-rings, and the dead volume (typically 55 defined as the residual space between each sample face and the windows) was reduced to about 0.4 ml by filling the empty space with KBr windows placed on each side of the sample holder. The photocatalytic system was connected to a flow set-up. Gases were introduced into the lines (heated at 333 K) by mass flow 60 controllers. The system allows the two gas mixtures, so called "activation" and "reaction" flows, to be prepared and sent independently to the reactor cell. Gases were introduced to the sample by 1/8" OD pipe and collected on the opposite side of the sample holder. More details can be found in references [36][37][38]. The low partial pressure of methanol was established using a saturator at controlled temperature. The gas mixture composition was then fixed at 1 vol. % methanol and 20 vol. % O2 diluted in

Results and discussion

The as-synthesized zeolite Beta and Ti-Beta composite obtained 85 by plasma treatment are characterized by XRD (Figure 1). Both display relatively broad Bragg reflections peaks characteristic of zeolite Beta structure, built of BEA and BEB polymorphs. No noticeable changes in the structure of the zeolite have been observed after plasma treatment. However, new broad peaks 90 appear in the two theta regions 23-26° and 45-50°. They correspond to TiO2 anatase phase formed on the Beta surface. In order to determine the textural properties of calcined Beta, Ti-Beta, TiO2/Beta and P25-TiO2 samples, nitrogen sorption analysis was performed. Table 1 summarizes the most important characteristics of these materials. As shown at Figure 3, all zeolite containing samples display a typical type I isotherm with hysteresis loops at high relative pressure. This latter is related to the textural porosity of aggregated nanosized zeolite crystals. The microporous volume was found to decrease in the following order Beta > TiO2/Beta > Ti-Beta. The specific surface area decreased as well in the same order 635, 570 and 420 m 2 g -1 for Beta, TiO2/Beta and Ti-Beta, respectively. The decreasing of the specific surface area and microporous volume of SSD prepared sample TiO2/Beta is obviously due to the presence of 15 wt % species ((SiO)3SiOH), respectively. The signal assigned to the ((SiO)3SiOTi) species, for Ti-Beta materials, appears at a higher field than that of (SiO)4Si) (Q4), which explain the low resolution of Q4-Q3 signals for this sample [41]. As shown at Figure 5, the 55 27 Al MAS NMR spectrum of Ti-Beta shows an increase of the signal at 0 ppm usually attributed to hexacoordinated aluminum species. Such a result reveals a partial dealumination of the structure after plasma treatment (in addition to the 5 wt.% of alumina extracted from the structure and detrmined by ICP 60 measurement). In addition, 1 Hà 29 Si cross polarization experiments (Figure 4B), that are more sensitive to Si-OH groups, show a spectacular increase of the Q4/Q3 ratio after the plasma incorporation of Ti. These results clearly point out a chemical interaction of Ti with the structure of zeolite and 65 suggest the formation of Si-O-Ti bond after TiCl4-plasma treatment. This process is certainly complex and involves the interaction of chlorine ions with Al species followed by formation of Si-O-Ti linkages. Unfortunately, it is not possible to quantify the number of Aluminum sites substituted since 70 hexacoordinated aluminium species are still present in the zeolite as shown by 27 Al mas NMR. But, according to ICP analysis this amount could be estimated to higher than 5%. Figure 6 includes the UV spectra of Beta-type material after calcination, Ti-Beta sample, and TiO2-p25. The initial Beta-type material exhibits a very low UV absorption. However, titaniummodified Beta material showed substantially different UV absorption characteristics, namely a blue shift of the UV spectra as compared to anatase and rutile forms of TiO2. A broad band is observed between 200 and 350 nm, with a maximum centered at ca. 270 nm (Figure 6). It is assigned to a charge transfer between oxygen and Ti atoms, simultaneously present in tetra-, penta-and hexacoordinated species. This band is broader than the one observed in TS-1 zeolite containing namely framework Ti [42,43]. Similar spectrum has been recorded for nanoclusters titanium oxide on silica [44]. Therefore, the UV spectra reveals the presence of clusters of titanium oxide in Ti-Beta zeolite [42-

. This result shows also the presence of species with different coordination, including tetrahedral coordination that could be attributed to Ti atoms in zeolite framework. The bright-field TEM image (Figure S3) shows Beta zeolite nanocrystal with typical size around 100 nm that are randomly surrounded by nanosized particles as it can be seen from at the 30 insert enlarged image. The insert ring ED pattern taken for this crystal shows evidence of coexistence of ultra small size TiO2 nanoparticles and Beta zeolite crystals. In particular, together with the diffraction spots attributed to Beta zeolite, a faint ring is observed in ED pattern. It corresponds to 3.5 Å which is typical (011) spacing of TiO2 anatase structure (ICSD 9852). This result is in agreement with that observed above by XRD. High resolution TEM (HRTEM) gives more evidence and information on the structure and spatial distribution of TiO2 nanoparticles. The bright field HRTEM image of single Beta nanocrystal reported at Figure 7 reveals a mixture of small and ultra small nanoparticles in the sample. The size and distribution of bigger particles is particularly clear in Figure 7a and7d. The small TiO2 particles have a typical size of 4-6 nm and definitely located at the surface of Beta nanocrystas. The surface of Zeolite nanocrystals is completely covered. Determine the structure and spatial distribution of clusters of TiO2 by TEM is far from trivial due to the overlapping of the layer of big TiO2 nanoparticles on the surface and zeolite Beta matrix. However, bright field HRTEM image in Figure 7c shows very small particles with a scale size of 1-2 nm eventually spread, as dark spots, throughout the specimen.Unfortunately it is not possible to determine the spatial position of Ti-O particles within Beta Zeolite framework due to the two dimensional aspect of TEM images, and even by using electron tomography because of extremely small size, week 20 contrast and complex structure of Ti-Beta material [45]. Nevertheless, the comparison of HRTEM images of pure Beta zeolite (Figure 7b) and Ti-Beta crystals highlights two important points. The first point concerns the crystal structure: Ti-Beta Zeolite crystals exhibit disordered lattice (Figure 7c) with respect 25 to pure Beta Zeolite crystals (Figure 7b). The second point is that the Ti-Ox nanoparticles appearing as dark contrast spots (marked by white arrows in Figure 2c) exhibit quite uniform size (~2 nm) and shape (square-like) distribution. As the Beta Zeolite consists of cubically arranged pores, it is logical to assume that the 30 nanoparticles should follow this arrangement if they are embedded within the structure framework. Thus according to these evidences, it is reasonable to conclude that cluster of titanium oxide are encapsulated within Beta Zeolite framework. In situ FTIR-plasma results: The IR spectrum of Beta zeolite sample displays two main narrow bands at 3740 cm -1 and 1630 cm -1 (Figure 8), in addition to the characteristic structural vibration bands. These two bands correspond respectively to the n(OH) of terminal silanol groups and to the d(H2O) of adsorbed water molecules [46][47][48]. The broad band appearing between 3700 and 2800 cm -1 is assigned to hydrogen bonded terminal silanol groups Si-OH with physisorbed water molecules [46][47][48].

The evolution of the IR spectra during plasma treatment and after each treatment step is reported at Figure 8-A and Figure 8-B, respectively. First, the introduction of TiCl4 leads to the disappearance of the band assigned to adsorbed water molecules in Beta zeolite. This is coupled with a decrease of the hydrogen bonding band related with the terminal silanol band. These changes in the IR spectra were assigned to the oxidation of TiCl4 to Ti(OH)nCl(4-n) (n≤3) in the presence of physisorbed water and to the formation of SiOTi(OH)mCl(3-m) (m ≤3) in the presence of terminal SiOH groups [47]. The total consumption of water molecules, traduced by the complete disappearance of the vibration band of water at 1630 cm -1 , shows that all the accessible sites to water were also accessible to TiCl4. The broad band of SiOH vibration, which is still present, is most probably a consequence of the hydrogen bonding between SiOH sites and hydrolysed titanium precursor. When TiCl4-plasma is turned on, a notable decrease of the SiOH bands is observed in the first few seconds. The observed changes are related to TiCl4 Silanol content in Ti-Beta and Beta samples: Figure 9 presents the IR spectra of zeolite Beta nanocrystals and Ti-Beta samples in the SiOH vibration region after activation of the parent zeolite 10 Beta samples at 623 K under vacuum for 1 h and after O2-plasma treatment for Ti-Beta. The silanol contents has been determined using the normalized area of (n+d)SiOH vibration band at 4570 cm -1 (insert of Figure 8, Table 1) [49]. An important decrease of SiOH groups after TiCl4-plasma followed by O2-plasma treatment can be observed (Figure 9, insert). The comparative study revealed that the relative silanol contents in Beta/Ti-Beta samples are equal to 2.4/1. This result unambiguously confirms that more than 50% of silanols defects disappear after TiCl4plasma treatment and proves that the extracted Aluminum species

20
were substituted by Ti. The analysis has been repeated several times in order to verify the reproducibility of the result and to estimate the relative incertitude (± 5%) on silanol ratio values. and 806 cm -1 . Another important and distinctive feature for Ti-Beta is observed at about 955 cm -1 . Although the assignment of this band is still in debate, the presence of this band after dehydration of samples under high-vacuum conditions is generally accepted as a solid proof for the incorporation of Ti into 40 the framework [41,[50][51][52][53]. This band is notably different from the one at 930 cm -1 that can be observed in zeolite Beta. The latter is assigned to the n(Si-OH) vibrations. In order to shed more light on the origin of this band the two samples were subjected to an isotopic H-D exchange. After D2O exchange, the 45 IR spectra of Ti-Beta and Beta samples (Figures 10-B) show a strong decrease of the bands in the n(OH) (3750-3500 cm -1 ) range with a concomitant formation of a n(OD) band (2760-2600 cm -1 ). Moreover a 20 cm -1 shift of the band at 930 cm -1 is also observed in the pure zeolite sample. This latter is attributed to the H-D 50 exchange of silanol groups [54]. A negligible shift, from 953 cm -1 to 955 cm -1 is observed in the case of plasma treated material. This result undoubtedly confirms the hypothesis of Si-O-Ti formation. the cluster form of titanium dioxide (Figure 11). These results are in a good agreement with the results obtained by EDS-SEM and HRTEM. On the other hand Py adsorption data do not point out dramatic modification in the acidic properties of Beta after plasma treatment.

Fig. 11 IR spectra of zeolite Beta, SSD prepared TiO2/Beta, Ti-Beta and P25-TiO2 samples after pyridine adsorption (spectra before Py adsorption have been subtracted).

Ti-Beta as photocatalyst:

In the present work, methanol photooxidation is used as a model reaction to study the photodegradation of volatile organic compounds (VOCs). No photocatalytic effect has been observed for Beta zeolite sample.

The conversion of methanol on Ti-Beta and P25-TiO2 are presented in Figure 12. Due to the different content of TiO2 in the two samples, methanol photodegradation rate (VMeOH) has been calculated. It corresponds to the amount of methanol converted (Vol.MeOH) per second per gram of TiO2 (g(TiO2)). VMeOH has been calculated with the following formula: VMeOH = [Vol.MeOH-converted (ml)]/[mTiO2 (g) x time (s)] (ml/ gTiO2/s). As shown at Figure 12, a close methanol conversion has been observed using both materials. The photodegradation rates show that VMeOH value obtained using Ti-Beta is 8 times higher than that obtained using P25-TiO2. This result could be assigned to the high dispersion and accessibility of titanium sites in Ti-Beta that compensate the low TiOx content in this sample. The higher performance could also be related to the effect of zeolite structure in the separation of the electron/hole charges formed under irradiation, which improves the photocatalytic activity. In addition, the well known acidic properties of Beta zeolite could increase the adsorption of organic compound and hence the photoreactivity.

On the other hand, the selectivity of P25-TiO2 and Ti-Beta photocatalysts was found to be different. More than 96% photocombustion was obtained in the case of P25-TiO2, whereas the formation of methyl formate, contributed to ~50% of the photooxidation on Ti-Beta catalysts. Thus the photocombustion 40 reaction contributed to ~50% of the overall transformation on Ti-Beta. As mentioned before the difference in the catalytic activity of bulk TiO2 is obviously related to the difference of active sites present in the two materials combined with the impact of zeolite structure in the case of Ti-Beta. These results allow to investigate 45 the use of Ti-Beta for green chemistry applications as the synthesis of methyl formate from methanol using light energy.

Conclusions

Low temperature TiCl4-plasma was used to incorporate highly 55 dispersed titanium species in the structure of zeolite Beta. The ultimate material was subjected to comprehensive characterization using complementary methods (XRD, NMR, SEM-EDS, IR, UV-visible, and N2 sorption). More than 50% of the silanol defects present in the zeolite structure were 60 disappeared after TiCl4-plasma treatment. The formation of chemical bonds "Si-O-Ti" was also revealed. Indeed, the increase of the hexacoordinated aluminum species accompanied with a decrease of the silanol defects pointed out the insertion of Ti into zeolite Beta framework. Pyridine adsorption showed higher 65 dispersion of TiOx in Ti-Beta sample, which was found 10 times higher than that of TiO2/Beta prepared by SSD method. The properties of zeolite Beta were not significantly affected after plasma treatment. The parent and the treated samples showed close acidic and adsorption properties. The elaborated Ti-Beta 70 composite was tested as photocatalyst for methanol photooxidation. Plasma prepared Ti-Beta catalysts showed 10 times higher methanol photodegradation rate in respect to the reference P25-TiO2 catalyst. This result demonstrated the potential of Ti-Beta photocatalysts for applications where the 75 temperature is a critical parameter. It also showed that Ti-Beta could be used for water or air purification, green synthesis or photovoltaic application. These results open the route to green photochemistry applications of Ti-containing zeolite catalysts prepared by cold plasma 80 method. Regarding the short time of the TiCl4-plasma treatment, in addition to its efficiency, cost effective and eco-friendly, this method is particularly attractive for commercialization. Furthermore it does not require the use of solvents. Fax: +33 2 31 45

Scheme 1 .

 1 Scheme 1. Plasma (A) and in situ FTIR-plasma (B) reactors.

65

  For this specific photocatalytic oxidation study, UV irradiation was carried out with a polychromatic light of a Xe-Hg lamp (LC8 spot light Hamamatsu, L10852, 200 W) with UV-intensity about 6 mW/cm 2 at l=366nm. It was performed using a UV-light guide (A10014-50-0110) fixed at the entrance of a modified operando 70 cell. The reaction temperature has been maintained at about 298 K.

75

  Ar and the total flow was adjusted to 25 cm 3 /min. The analysis of the outlet gases was performed by means of a Pfeiffer Omnistar mass spectrometer. P25-TiO2 has been used as reference in the same condition. Methanol conversion and CO2-selectivity has been calculated using the m/z=31 and m/z=44 MS signals, 80 respectively [37].

4 |

 4 Journal Name, [year], [vol], 00-00 This journal is © The Royal Society of Chemistry [year]

Fig. 1

 1 Fig. 1 XRD of pure anataseTiO2 (a), Beta zeolite (b) and Ti-Beta (c) samples. Note: *Peak assigned to anatase phase.

Figure 2 ,

 2 presents the EDS spectrum of Ti-Beta sample. It displays two peaks at 4.5 eV and 4.8 eV. According to the literature, these two peaks are assigned to Ka1 and Kb of titanium, respectively [40]. According to ICP results, the Si/Al ratio increases of about 5% after plasma treatment from Si/Al=20.1 to 21.2. This result shows a partial extraction of Al from zeolite structure after plasma treatment. The amount of Titanium species introduced is estimated to 11 wt %. Besides, the cartography measurements performed on the composite Ti-Beta sample and the mechanical mixture TiO2/Beta (15/85 % wt.) show clearly the higher dispersion of Ti atoms in the Ti-Beta structure in respect to TiO2/Beta (Figure S1 in supplementary information).

Fig. 2

 2 Fig.2 EDS spectrum of Ti-Beta sample.

35 bulkFig. 3

 353 Fig. 3 Nitrogen adsorption (close symbol) and desorption (open symbol)45

Figure 4

 4 Figure 4 shows the 29 Si MAS NMR spectra of the initial zeolite Beta and Ti-Beta samples. The two main signals, observed at50

Fig. 4 29

 4 Fig. 4 29 Si MAS NMR (a) and CP MAS 1 Hà 29 Si (b) NMR spectra of Beta and Ti-Beta samples.

Fig. 5 27

 5 Fig. 5 27 Al MAS NMR spectra of Beta and Ti-Beta samples.

Fig. 6 25 (

 625 Fig.6 DR UV-visible spectra of zeolite Beta (a), Ti-Beta (b) and P25-TiO225

6 |

 6 Journal Name, [year], [vol], 00-00 This journal is © The Royal Society of Chemistry [year]

Fig. 7 a

 7 Fig.7 a) Bright field HRTEM image of single Ti-Beta Zeolite crystal; b) bright field HRTEM image of parent Beta zeolite; c) Enlarged HRTEM image of Ti-Beta. The dark spots Ti-O nanoparticles are pointed by white arrows; d) Enlarged image of the region marked in (a). The TiO2 particles with range size between 4-6 nm are depicted by white arrows.

65 7 Fig. 8 (

 78 Fig.8 (A) Evolution of zeolite Beta IR spectrum vs the treatment time: during TiCl4 introduction (a), during TiCl4-plasma (b) and during O2plasma treatment (c). (B) IR spectra of Beta zeolite (i), after TiCl4 introduction (ii), after TiCl4-plasma (iii) and after O2-plasma (iv) treatment.

Fig. 9

 9 Fig.9IR spectra of Beta sample after activation at 673 K under vacuum (a) and Ti-Beta sample after plasma treatment in the OH vibration region (b). Spectra are normalized to the same weight of pellet.

55 Fig. 10 ( 8 |

 55108 Fig. 10 (A) IR spectra of Ti-Beta (a) and initial zeolite Beta (b) calcined samples diluted in KBr (2 wt.%). (B) IR spectra of Ti-BEA (a) and Beta (b) calcined samples diluted in KBr (5 wt.%) before (1) and after (2) D2O exchange.60

Fig. 12

 12 Fig.12 Methanol conversion and CO2-selectivity during methanol

85Methylformate

  Notes and referencesa Laboratoire Catalyse et Spectrochimie, Université de Caen Basse-Normandie, ENSICAEN CNRS, 6 bd Maréchal Juin, 14050 Caen, France b Laboratoire de Crystallographie et Science des Matériaux, Université de Caen Basse-Normandie, ENSICAEN CNRS, 6 bd Maréchal Juin, This journal is © The Royal Society of Chemistry [year] Journal Name, [year], [vol], 00-00 | 9

  28 22; Tel: +33 2 31 46 27 32; E-mail: mohamad.elroz@ensicaen.fr † Electronic Supplementary Information (ESI) available: cartographycal analysis of Si and Ti in Ti-Beta and SSD TiO2/Beta samples. Evolution of the IR spectra of Beta and Ti-Beta during Py desorption under vacuum at different temperature. Bright field TEM image showing Beta Zeolite -TiO2 crystals and the corresponding ED pattern. See DOI: 10.1039/b000000x/ 1 D.F. Ollis, E. Pelizzetti, N. Serpone, Environ. Sci. Technol. 1991, 25,

Journal Name, [year], [vol], 00-00 | 3