Hajime Fukui

Wei-Yan Chow

Jing Xie

Vivek Vasudevan

Choon Hwai Yap

Nicolas Minc

Naoki Mochizuki

Julien Vermot

Molecular control of endocardial cell fate in response to mechanical forces during cardiac valve formation

Developing cardiovascular systems use mechanical forces at all organizational scales to shape tissue. How ubiquitous blood flow forces instruct local cardiac cell identity is still unclear. By manipulating mechanical forces in vivo, we show that ectopic shear stress is necessary and sufficient to promote ectopic valve like structure by activating cellular valve identity and Endothelial to Mesenchymal Transition. We found ectopic valve formation results from the activation of two mechanosensitive pathways acting in parallel, the well-established TRP-klf2a-wnt9b axis and an extracellular ATP-dependent purinergic receptor pathway specifically triggering Ca2+ oscillation and Nfatc1 activation. Thus, mechanical forces are converted into discrete bioelectric signals by synergistic mechanosensitive pathways to generate positional information and control valve formation.

Main Text:

The cardiovascular system is continuously exposed to mechanical forces, such as fluid shear stresses and stretching forces generated by blood flow and heartbeat. The mechanotransduction cascade translates forces into cellular biochemical signals in the cell (1,2). The lumen of the cardiovascular system is lined by endocardial cells (EdCs) and endothelial cells (ECs). These cell types present unique functional properties and differentially respond to forces depending on the type of blood vessels or heart regions they belong to (3)(4)[START_REF] Ranade | Proc. Natl. Acad. Sci. U. S. A[END_REF][START_REF] Xu | [END_REF]. Heart valve defects are common human congenital anomalies, with an incidence close to 2% of live births (7). While a number of valvular heart defects have been linked to specific genetic mutations (e.g., in NOTCH1, TBX5, GATA4, TBX20, LMCD1, TNS1, and DCHS1) (8)(9)(10)(11)(12), the majority have no clear environmental or genetical cause (13). Thus, it is thought that non genetically encoded factors play an essential role in the genesis of congenital valve defects. The forces generated in the heart are significantly higher than in the vascular system where pathologies related to mechanical forces start to be well established (14,15). Consequently, the molecular mechanisms underlying force sensitivity of endothelial cells in the heart (i.e. EdCs) are likely to be different from vascular endothelial cells and remain largely unknown. Calcium ion (Ca 2+ ) signaling is often associated with the direct cellular response to mechanical forces and initiates the mechanosensitive response in endothelial cells (16). Yet, how Ca 2+ signaling leads to a cell specific response to mechanical forces remains unclear. Here, we examine the ability of mechanical forces to modulate specific endocardial fate towards valvulogenesis. We directly tested the signaling cascades activated by mechanical forces, quantitatively characterized their dynamic properties, and considered the implications of these observations for transcriptional regulation of valve-specific markers in vivo. We find that valvular cell identity is directed by blood flow via two different mechanosensitive pathways: TRP mediated klf2a activation and ATP mediated Ca2+ oscillation leading to Nfatc1 signaling in response to local high shear stresses.

Using the zebrafish model system to allow for high precision control of spatiotemporal mechanical parameters (17,18), we analyzed Ca 2+ dynamic patterns via live imaging of the endocardium at cellular resolution. Ca 2+ fluctuations are observed in many developmental contexts associated with tissue reorganization such as cell migration (19), wound healing (20), embryonic development, and regeneration (21,22). In endothelial cells, Ca 2+ fluxes have been shown to be involved in angiogenesis (23,24). Thus, it is thought that Ca 2+ is an important effector of cardiovascular morphogenesis. To address whether Ca 2+ activation can be observed specifically in the EdCs, we expressed the fluorescent Ca 2+ sensor protein GCaMP7a in endocardial/endothelial cells using a Tg(fli1a:gal4ff);(uas:GCaMP7a) system (24) along with Tg(kdrl:nls-mCherry). We found that Ca 2+ oscillations were observed almost exclusively in the EdCs of the atrioventricular canal (AVC) (Fig. 1, A to D, Fig. S1A and Movie S1) between 32 and 102 hpf (hours post fertilization). Normalization of the calcium signal obtained by computing the ratiometric intensity of GCaMP7a to nls-mCherry signals in tracked EdCs confirms that EdCs display Ca 2+ oscillations (Fig. 1B and Movie S2). The occurrence of Ca 2+ oscillations peaks at around 2.5 dpf and subsequently declines as the morphogenesis of functional valves proceeds (Fig. 1C). We found that around two thirds of EdCs in the AVC display Ca 2+ oscillating at 54 hpf, and that this number decreases at later developmental stages (Fig. S1B). Similarly, the oscillation frequency reaches a maximum at 54 hpf and declines at later stages (Fig. S1C). Interestingly, we found that the period of Ca 2+ activation does not significantly vary with development stages even when the overall activity declines (Fig. S1D).

To verify that the calcium oscillations are associated with valvulogenic differentiation, we next searched for the decoder/effector of this Ca 2+ signaling. Nuclear factor of activated T-cells (Nfatc1) is an established Ca 2+ sensitive transcription factor known to control EndoMT and subsequent heart valve morphogenesis (25)(26)(27)(28)(29)(30). The inactive form of Nfat is phosphorylated and sequestered in the cytoplasm. Nfatc1 activation is mediated by calcium via the calmodulin dependent serine/threonine protein phosphatase Calcineurin in response to Ca 2+ (43). Upon the activation of Calcineurin, Nfat is dephosphorylated and subsequently translocated to the nucleus to activate transcription (30,31). Nfatc1 is thus both a good marker of valve cell identity and a candidate for a relay protein decoding the levels of cytoplasmic Ca 2+ to generate a transcriptional response.

(Fig. S2A). We generated a Nfat binding element reporter line, Tg(4xnfbr:d2EGFP), which expresses d2EGFP in response to the binding of nuclear-localized Nfat protein (Fig. S2B). Nfat reporter expression was observed in EdCs of the AVC from 34 hpf onward (Fig. 1, E and F, and Fig. S2C). At 78 hpf, the number of d2EGFP positive cells starts to decrease in the EdCs of AVC, but the reporter still displays a specific expression in EdCs in the AVC (Fig. 1E). Nfat reporter activation was thus strikingly similar to the time course observed for the Ca 2+ response (Fig. 1, C and F). To confirm these results, we additionally tracked Nfat nuclear re-localization by generating an endothelial/endocardial-specific GFP-Nfatc1 reporter allowing to monitor Nfat subcellular distribution (Fig. S2F). As expected, we found that the GFP-Nfatc1 localisation varies in the endocardium with strong nuclear localization in the AVC while GFP-Nfatc1 remains cytoplasmic in the other cells of the endocardium (Fig. S2F).

Together, these results highlight that both spatial and temporal regulation of Ca 2+

signaling and Nfat activation are unique features of the EdCs located in the developing valve area during heart development, suggesting an important role during valve formation in response to mechanical forces.

Previous studies have shown that Ca 2+ levels in EdCs are associated with changes in mechanical forces but Ca 2+ dynamics in response to mechanical force changes have never been described (4). To test if the observed Ca 2+ oscillations in EdCs are force-responsive, we first assessed the Ca 2+ signals in hearts stopped using of MS-222 or p-amino blebbistatin treatment. We found that in these conditions, Ca 2+ activation was abrogated in EdCs (Fig. S3, A to C). Importantly, Ca 2+ oscillations recovered upon wash-out and restarting of the heartbeat (Fig. S3, A and B). Next, we assessed the impact of decreased flow forces associated with lower hematocrit by analyzing the gata1a mutant zebrafish line that do not have red blood cells (32)[START_REF] Lyons | Proc. Natl. Acad. Sci. U. S. A[END_REF][START_REF] Vermot | [END_REF]. Lower hematocrit significantly affects shear stress and flow profile in the AVC (35,36). Consistent with the heart-arrested condition, gata1a mutants (and gata1a morphants), exhibited a decreased number of Ca 2+ spikes at both 54 hpf and 76 hpf (Fig. S3, D andE). We also found a significant decrease of Nfat activity in the gata1a mutants (Fig. S3F). Together, these results suggest that both Ca 2+ oscillations and Nfat activation are stimulated in response to mechanical forces generated by the beating heart.

In the heart, cardiomyocytes have been proposed to activate EdC responses to forces, suggesting that endocardial cells are not the direct force sensor (37). It is thus important to confirm that mechanical forces are indeed directly leading to Ca 2+ signaling in the endocardium. Consequently, we set out to alter the mechanical force distribution in the living heart by artificially changing the boundary conditions and thus the flow forces in the system (Fig. 2A). To this end, we inserted single agarose-based magnetic beads into the cardiac lumen by microsurgery using 30-60 m diameter beads that can be inserted in the atrium or the ventricle without completely blocking heart function and flow in the beating heart (Fig. 2B and Movie S3). The presence of the bead in the heart alters the flow forces generated in the beating heart. To quantify the flow generated around the bead, we performed a 3D simulation of the wall shear stress and pressure around the bead using image-based computational fluid dynamics simulation with a dynamic wall motion (38) (Fig. 2C and Fig. S4, A to C). We found that both parameters are increased in the vicinity of the bead, indicating an increase in the flow forces generated around the grafted bead (Movie S4). Interestingly, the calculated wall shear stress amplitude generated by the presence of the bead in the atrium (3 Pa) is close to the endogenous wall shear stress amplitude generated in the AVC at 48 hpf where the valve normally forms (between 3 and 7 Pa) (4,38,39). In vivo, we analysed the Ca 2+ response of the EdCs and found that Ca 2+ spikes were observed in the EdCs close to the bead when trapped in the atrium or in the ventricle, whereas sham injected embryos never display any Ca 2+ activation except in the AVC (Fig. 2D, Fig. S4, D and E, and Movie S5 and S6). The Ca 2+ activation period lasted an average of 5.5 sec around beads in the EdCs of the atrium or the ventricle (Fig. S4F).

To further demonstrate that the impact of shear stress directly affects the EdC calcium response in live embryo, we directly manipulated the bead within the heart using magnetic tweezers. This approach permits to generate controlled shear forces in an immobilized heart (Fig. 2E). We first calibrated the velocity of the bead pulled by the magnetic tweezer and quantified the amount of force it can generate in the zebrafish heart (Fig. S4, G andH). We found that the force generated by the magnetic bead in motion ranged between 100 to 2000 pN, a stimulus which is slightly higher than endogenous wall shear stresses generated in the AVC (4,38,39). While the magnet probe alone did not affect Ca 2+ influx in EdCs, direct pulling of the magnetic bead lead to the generation of Ca 2+ influx in the EdCs of the arrested heart (Fig. 2, F to H, Fig. S4, I and J, and Movie S7). Together, these results indicate that Ca 2+ fluxes are induced in the EdCs by mechanical forces such as wall shear stress and associated wall pressure.

We next assessed the biological relevance of the localized response initiated by bead grafting and tested the role of ectopic forces in the initiation of valve formation.

Considering that bead grafting leads to the generation of a typical mechanosensitive response in ectopic areas of the heart, we addressed if it is associated with the formation of ectopic valves. Notably, 16-20 hours after bead grafting in the atrium, valve-like EdCs clusters were observed near the grafted beads. The valve like structures were visible in the atrial region that protruded from the atrial surface to the atrial cavity or the cardiac jelly, respectively (Fig. 3A). We found that both the activity of Nfat and Notch signaling, which are specific to valve cell identity (40)(41)(42), were strongly activated in the valve like EdCs cluster generated near the grafted bead (Fig. 3A and Fig. S5A). To confirm the results, we grafted a bead into atrial lumen at 50 hpf or 74 hpf and assessed the Nfat reporter activity as a response to increase forces. Four hours after bead grafting, Nfat reporter response was ectopically activated in EdCs located near the bead (Fig. 3B and Fig. S5B). These results were consistent with the fact that Nfat activity is dramatically reduced when heart contractility is affected (Fig. S5, C andD). Conversely, we found that Nfatc1 nuclear localization is activated in response to ectopic forces (Fig. S5E). We could confirm that nuclear activity of nfatc1 is dependent on the ser/thr de-phosphorylation by using a chemical blocker of Calcineurin (FK506) (Fig. S5E). Thus, the nuclear localization of GFP-Nfatc1 is promoted by increased mechanical force, in a Nfatc1 ser/thr de-phosphorylation dependent manner.

To clarify the cell identities within the ectopic structures, we followed two different approaches. We first generated a BAC transgenic line using the well-established EndoMT marker twist1b, TgBAC(twist1b:GFP), to highlight cells that underwent EndoMT in the valvuloids. We clearly found Twist1b positive cells undergoing EndoMT in the developing valve cushion and strong expression in the mesenchymal cells of the developing valve (Fig. S6, A to C). When ectopic forces generated by beads lead to valvuloid structures, Twist1b positive cells were populating the valve-like structure (Fig. 3C). We also observed that the grafted beads trigger the generation of mis-localised protrusions into cardiac jelly, which constitute the first sign of cardiac valve morphogenesis in zebrafish (44) (Fig. 3A). This observation along with the presence of Nfat and Notch positive cells indicate that the ectopic valve like structures have important similarities with endogenous valves.

To further confirm these observations, we next assessed if other valve related genes are induced in response to the presence of ectopic forces in the endocardial cells (Fig. S6,D to H). Klf2 and Wnt9b have been shown to control heart valve remodeling in response to mechanical forces in mouse and zebrafish (45). Using RNAscope, we found that the grafted beads induced the ectopic expression of klf2a and wnt9b in the endocardial cells surrounding it. In addition, we found that klf4a, a known modulator of EndoMT in EC (46,47) was also activated in response to ectopic forces. Interestingly, both klf2a and klf4a expression were not affected when calcineurin was inhibited FK506, suggesting that Ca 2+ -Nfat signaling pathway act independently of the klf2a-wnt9b mechano-transduction pathway. Conversely, egr1, a known mechanosensitive gene specifically expressed in the AVC (48), was downregulated by calcineurin inhibition (Fig. S6, I andJ). This suggests that egr1 acts downstream of Nfatc1. Thus, endocardial cells experiencing ectopic forces activate the morphogenetic program which is promoting the expression of valve remodeling and EndoMT markers required for the endogenous AV valve development.

These results demonstrate that mechanical forces are key to specify heart valve position during development by modulating both the Klf2a-Wnt9b and the Nfatc1 signaling pathways.

Considering Nfat and Klf2a-Wnt9b seem to define two different mechanosensitive pathways, we next characterized the factors which are responsible for the force-dependent Ca 2+ -Nfat signal activation. We first focused on stretch sensitive channel proteins (3,4) and primary cilia (49) because they have been widely implicated in flow sensing in the cardiovascular system (3,50). We used established mutant lines for the stretch sensitive channels Trpp2, Trpv4, Piezo1, Piezo2a and Piezo2b (4, 51, 52) and iguana (Dzip1) (53) which is responsible for ciliogenesis in the cardiovascular system (54). We found that all of these mutants showed normal Ca 2+ activation (Fig. S7, A andB). These results were confirmed by the fact that gadolinium ion (Gd 3+ ), a non-specific stretch-sensitive channel blocker, did not suppress the Ca 2+ influx significantly (Fig. S7C). These results suggest that primary cilia and stretch sensitive channels are not the main regulators of the Ca 2+ oscillations observed in EdCs.

Considering none of the expected stretch sensitive channels affected calcium oscillations, we turned to paracrine signals potentially released in response to mechanical forces.

Previous studies have shown that Adenosine 5' triphosphate (ATP) is released in cultured endothelial cells in response to mechanical stimuli (16). As a response, ATP activates Ca 2+ signaling via an increase of the permeability of cations in purinergic receptor P2X channels (55) (Fig. 4A). We thus hypothesized that intracellular Ca 2+ fluxes are dependent on ATP levels. ATP can be generated by endothelial mitochondria (56). Accordingly, we found that the Ca 2+ influx in EdCs of the AVC was abolished when cytosolic ATP levels are depleted by oligomycin A, a mitochondrial ATP synthase inhibitor (Fig. S7D). We next investigated whether extracellular ATP is able to alter Ca 2+ influx in EdCs by microinjecting oligomycin A, apyrase (an ATPase) and ATPS (an ATP analog) into the cardiac lumen (Fig. 4B, and Movie S8). After both oligomycin A and apyrase injection, we found that the Ca 2+ influx was significantly suppressed in the EdCs of the AVC; conversely, ATPS significantly increased the amount of Ca 2+ influx (Fig. 4C, and Fig.

S7, E to G). In addition, ATPS led to a significant increase of both the number of cells displaying Ca 2+ spikes and the Ca 2+ oscillation frequency in the EdCs (Fig. S7, H andI).

Importantly, the heart rate and the cell viability were not affected by these treatments, suggesting that none of them altered the mechanical forces associated with heart function.

These results indicate that extracellular ATP is the trigger of Ca 2+ signaling in EdCs in response to flow.

The requirement of extracellular ATP for the Ca 2+ influx in EdCs prompted us to investigate the involvement of known ATP dependent channels such as P2X channels that are activated by disturbed flows in response to ATP release in endothelial cells (57,58).

We first examined whether P2X (P2rx in zebrafish) channels are expressed in the EdCs.

We found that p2rx1, p2rx4a and p2rx7 transcripts are present in the endocardial cells of the heart at 48 hpf and 72 hpf (Fig. S8, A andB). Thus P2rx expression is not restricted to the AVC. Treatment against antagonists of P2X1, 4 and 7 decreased the number of Ca 2+ spikes in the EdCs (Fig. S8C). In line with this result, the embryos overexpressed a dominant-negative form of either human P2X4 (hP2X4C353W) or zebrafish p2rx4a (zP2rx4aC365W) mRNA, as well as p2rx4a morphants, displayed a significant decrease in the number of Ca 2+ spikes in EdCs without affecting heartbeat (Fig. 4D, and Fig. S8, D to G, and I). Overall, these results show that the Ca 2+ influx in EdCs is modulated by P2X channels in response to changes in extracellular ATP levels.

To understand how the activation and inhibition of P2X-mediated ATP signaling could affect valve formation, we first assessed how altering calcium oscillations translate into changes in gene expression. We tested whether P2X channels are involved in modulating Nfat activity in EdCs. Although embryos treated with combinations of P2X1, 4 and 7

antagonists, combinations of P2X4 with P2Y2 antagonists, P2X4 antagonist, or P2Y2 antagonist significantly suppressed the Nfat reporter activation, the other P2X/P2Y

antagonists had no significant effect (Fig. S9, A andB). Furthermore, injection of a dominant-negative form of p2rx4 mRNA significantly suppressed reporter expression (Fig. 4E and Fig. S8H), which correlates with abnormal Ca 2+ activation (Fig. 4D and Fig. S8G). Although Vegf signaling is known to activate Nfat signaling (59), Ki8751, a Vegfr2

inhibitor, had no effect on both Nfat reporter expression and Ca 2+ activation in the EdCs (Fig. S9, A to D). Together, these results suggest that P2X and P2Y receptors coordinately modulate Nfat activity in the EdCs independently of VEGF signaling. In addition, we found that the Nfat activity in the endocardium seems unrelated to the tyrosine phosphorylation of Nfat determined by treatment of Janex-1 inhibiting jak3 which specifically phosphorylates Nfat tyrosine (Fig. S9, A andE).

We finally tested the valve phenotype in the embryos with suppressed extracellular ATP signaling. Although we found that Twist1b-positive cells localized in the endocardium, they were mis-localized when p2rx function was inhibited (Fig. 4F and Fig. S10, A andB). In addition, embryos displayed abnormal valve structures with incomplete morphogenesis of the valve and absence of leaflet formation (Fig. 4G). We additionally found that endocardial cells were excluded from the luminal part of the valve when Nfat activity is suppressed in these cells (Fig. S10, C andD). Altogether these results confirm that P2X functions acts upstream of Nfat activity to control EndoMT necessary for the valve development.

Mechanical forces have been implicated in a broad range of events during embryonic development (60)(61)(62). The presented work reframes our view of how endothelial cells interpret mechanical forces and supports the intriguing possibility that valve formation is mainly defined by the mechanosensitive inputs resulting from the local tissue constraints and flow forces (26,44,(63)(64)(65) Such treatment also led to the suppression of reporter activation (Fig. S2E), demonstrating that our reporter Tg(4xnfbr:d2EGFP) is indeed revealing Nfat activation. The following zebrafish mutants were used for experiments: The knockout alleles of m651 for gata1a [START_REF] Lyons | Proc. Natl. Acad. Sci. U. S. A[END_REF], ts294e for dzip1 (53), tc321 for trpp2 (52), sa1671 for trpv4 (4) (Zebrafish International Resource Center [ZIRC]), sa12608 for piezo1 (European Zebrafish

Resource Center [EZRC]), sa12414 for piezo2a (ZIRC), sa14724 for piezo2b (ZIRC).

The knockout allele of ncvxx for p2rx1 and ncvxx for p2rx4a were generated as described in the experimental procedures.

Generation of knockout zebrafish by TALEN mutagenesis

To develop knockout zebrafish, we used transcription activator-like effector nuclease Embryos then were released from agarose and allowed to develop in E3 medium at 28.5 o C.

Approximately 30% embryos kept the bead move continuously in the atrium or the ventricle together with the blood flow were applied to the experiments. Whereas, the embryos with the bead sucked in the outflow tract were excluded for the experiments.

Simulation of influences in the grafted bead in the atrium

A static geometry of the zebrafish atrium was approximated from a 2D fluorescent imaging video of the atrial chamber containing the moving magnetic bead. 2D outlines of the atrial wall and AVC were extracted from the video, imported in ANSYS Workbench 2019 (ANSYS Inc.) and revolved about their respective central axes to generate a 3D shape of the atrial chamber and AVC (Fig. S3A). A 30 m spherical bead was inserted in the chamber. The atrial chamber was meshed with about 453k tetrahedral cells, and a zone of 30 m radius from the center of the bead was programmed to have an overset mesh with 384k tetrahedral cells, which were determined by a mesh convergence study. In both meshes, refined mesh at wall boundaries was achieved via 5-10 inflation layers. The overset mesh was superimposed onto the atrial chamber mesh, and together with the bead, was programmed to move with a uni-directional oscillatory motion via ANSYS user-defined function. The bead motion waveform (Fig. S3B) was traced from the 2D video mentioned above. The bead motion was stopped at about 0.44 m from the atrial wall to avoid numerical problems as an estimation of the impingement. The blood fluid in the atrial chamber was assumed to have a viscosity of 3.5 cP and density of 1060 kg/m 3 . The inlet and outlet boundary conditions were set to zero reference pressures. Two cardiac cycle (800 time-steps) was simulated, with results were extracted from the second cycle. In another variant of the simulation, flow velocities were estimated from the inlet boundary via blood cell motions in the video and were imposed as inlet flow boundary conditions to give a more realistic boundary condition, but the results did not vary significant, and this was thus not considered. The limitation of the simulation was that there were no consideration of the motion of the atrial walls, however, given that the bead motion velocity was higher order than atrial wall motion velocity, our simulations' estimate of the peak wall shear stress and pressures at the bead impact location on the atrial wall should still be reasonable reliable.

Magnetic tweezer

A magnetic tweezer was used to apply force to magnetic beads (PureCube Glutathione where V is the velocity of the magnetic bead and x is the distance between the bead and the magnet tip. V300 is the bead velocity at a distance 300 m away from the magnet tip.

The same experiment performed on agarose magnetic beads with varying sizes at a fixed distance of 300 m then allowed to compute the net magnetic force, and establish force-size relationships, using the Stokes equation.

𝐹 300 = 0.1533 * 𝑅 3 where R is the bead radium (m). F300 is the magnetic force (pN) at a fix distance 300 m from the magnet tip.

According to velocity-distance and force-size relationships, we computed the final equation for calculating the magnetic force:

𝐹 = 0.1533 * 𝑅 3 * 𝑉 𝑥 𝑉 300
where F is the magnetic force (pN), and Vx (m/s) is the velocity at x m in the magnetic field. Pressure and shear stresses were deduced by projecting the forces, orthogonal or parallel to the tissue plane, respectively, and by dividing them a measured contact surface area between the bead and the tissue.

Microinjection into cardiac lumen

Tg(fli1a:galff);Tg(uas:GCaMP7a);Tg(kdrl:nls-mCherry) embryos were kept lateral side and embedded into 1% low-melting agarose. After mounted, we injected with 2 nL of apyrase (0.9 U/L, Sigma-Aldrich), ATPS tetralithium salt (45 mM, Tocris), and Oligomycin A (25 g/mL) together with red fluorescent 0.5 m Fluospheres (Thermo Fisher Scientific) using Nanoject II (Drummond) into the region of common cardinal vein.

To confirm the precise injection, circulation of Fluospheres in cardiac lumen was checked by the fluorescent stereo zoom microscope (Leica MZ FLIII), and a lightsheet microscope was used to evaluate the Ca 2+ influx. pA plasmid. The fragment of VIVIT-mCherry-pA site is inserted into pTol2-crystallin-Nls-mCherry-5xuas vector to establish the pTol2-crystallin-Nls-mCherry-5xuas-VIVIT-mCherry-pA plasmid.

To monitor the EndoMT, we established a GFP reporter transgenic line under the control of twist1b BAC enhancer/promoter; TgBAC(twist1b:GFP) ncv534 . pRedET plasmid (Gene Bridges, Heidelberg, Germany) was introduced into E. coli containing CH73-181M7 BAC clone encoding twist1b gene (BacPAC resources) by electroporation (1800V, 25 mF, 200 Ω). Tol2 long terminal repeats in opposite directions flanking ampicillin resistance cassette were amplified by PCR using Tol2_amp as a template and were inserted into the BAC vector backbone. The cDNA encoding GFP together with a kanamycin resistance cassette (GFP_KanR) was amplified by PCR using pCS2-GFP_KanR plasmid as a template and inserted into the start ATG of the twist1b gene. Primers to amplify the GFP_KanR for twist1b were 5'-atgctggaataacgtccttattcgcacgcgctttcagcagagacttaagcACCATGGTGAGCAAGGGCGAG GAG-3' and 5'-tccgcgggagacacgggggagctggaggagtctcgcgcgggctcttcgggTCAGAAGAACTCGTCAAG AAGGCG-3' (small letters; homology arm to BAC vector, and capital letters; primer binding site to the template plasmid).

To monitor the subcellular localization of Nfatc1 in the EdCs, we established a transgenic line expressing GFP-tagged Nfatc1 under the control of fli1a promoter; Tg(fli1a:GFP-Nfatc1) ncv532 . Primers to amplify the human-Nfatc1 (hNfatc1) were (5'-agatctCCAAGCACCAGCTTTCCAGTCCCTTC-3' [containing BglII sequence] and 5'-gtcgacCAGTTAAATGTGCAACACGCCAC-3' [containing SalI sequence]). The construct was inserted into the pEGFP-C1 vector to establish pEGFP-hNfatc1. The fixed with 4% PFA for 20 min at room temperature and subsequently immersed in glycerol. Images were taken using a stereo microscope (Leica M165C).

RNAscope analysis

Embryos were fixed by 10% NBF for overnight at room temperature. After fixation, the solution was changed to 50% Methanol / PBBT (PBS with 2 mg/mL BSA and 0.1% TritonX-100) for 10 min, then changed to 100% Methanol at room temperature, and then stored in 100% Methanol at -20 o C overnight. After rehydration, embryos were washed three-times for 10 min in PBBT. Embryos were treated in PBBT with protease plus for 30 min at room temperature, and subsequently washed three-times for 10 min in PBBT.

For the probe hybridization, embryos were treated in the following probe mixture (ACDbio): kdrl-C1 probe with p2rx1-C3, p2rx4a-C3 or p2rx7-C3 probes; klf4a-C1 probe with klf2a-C2 probe; wnt9b-C1 probe with klf2a-C2 probe; kdrl-C1 probe with gata1a-

Figures and legendsFig. 1 .

 1 Figures and legends

Fig. 2 .

 2 Fig. 2. Force directly triggers Ca 2+ influx in EdCs of zebrafish heart. (A)

Fig. 3 .

 3 Fig. 3. Artificial forces lead to the formation of valve like structures. (A)

Fig. 4 .

 4 Fig. 4. Extracellular ATP and P2rx channels function proper valve development. (A) Schematic of Ca 2+ influx via extracellular ATP-dependent P2XRs activation. (B) Schematic of microinjection into cardiac lumen. (C) The number of Ca 2+ spikes in EdCs

Fig. S2 .

 S2 Fig. S2. Characterization of Nfat reporter during heart valve development. (A) Schematic of Nfat signal activation via Ca 2+ influx in EdCs. (B) Schematic of the reporter

Fig. S3 .

 S3 Fig. S3. Heartbeat and fluid shear forces control the Ca 2+ influx in EdCs of the AVC.

Fig. S4 .

 S4 Fig. S4. Retention of a bead in the cardiac lumen exerts forces on EdCs. (A)

Fig. S5 .

 S5 Fig. S5. Bead retention leads to the ectopic reporter activation. (A) Representative

Fig. S6 .

 S6 Fig. S6. Nfat activation is dispensable for the force-dependent klf2a/klf4a expression.

Fig. S7 .= 7 ;

 S77 Fig. S7. Ca 2+ influx in EdCs is regulated by extracellular ATP-dependent signaling.

Fig. S8 .

 S8 Fig. S8. Ca 2+ influx and Nfat reporter activation are regulated by P2rx channels

Fig. S9 .

 S9 Fig. S9. Several P2Xs/P2Ys coordinately regulate Nfat activity in EdCs. (A) Violin

Fig. S10 .

 S10 Fig. S10. Nfat-suppressed EdCs are excluded for the luminal area of the AVC. (A) Boxplot representing the number of twist1b + EdCs of the AVC in embryos injected with

(

  TALEN) Targeter 2.0 (https://tale-nt.cac.cornell.edu) to design TALEN pair that targets p2rx1 and p2rx4a. The target sequence of TAL-p2rx1 and TAL-p2rx4a were 5'-TGCCATCGGCTCAGTCTTCACcaagatgaagggagtgTCCTTCACTAATATCAGTGGA -3', and 5'-TTCTCAGCTCAGTCACaaccaaagtgaaaggAATTGCTTTAACAAACACCA -3', respectively (capital letters were sequences of left [TAL-p2rx1-F and TAL-p2rx4a-F] and right [TAL-p2rx1-R and p2rx4a-R] arms, respectively). These expression plasmids of the TALEN-pair were constructed by pT3TS-GoldyTALEN. TALEN mRNAs were synthesized in vitro by T3 mMessage mMACHINE kit (Thermo Fisher Scientific). To induce double strand breaks in the target sequence, both 50 pg of TAL-p2rx1-F / -p2rx1-R mRNAs, and TAL-p2rx4a-F / -p2rx4a-R mRNAs were injected into 1-2-cell stage transgenic embryos, respectively. Each injected founder (F0) fish was outcrossed to wild-type fish to obtain the F1 progeny from individual founders. To analyse TALEN induced mutations, genomic DNA from F1 embryos was lysed by 50 l region is defined by the area located between the superior and inferior region of the atrium and the ventricular chamber. Images were processed with a Las X software (Leica), Zen 3.1 software (Zeiss), FV31S-SW software (Olympus), ImageJ and Imaris 9.6 (Bitplane).Chemical treatmentsEmbryos were treated with the following small molecule inhibitors: MS-222 (2.0 mg/mL) or BDM (2,3-butanedione monoxime, 30 mM, Sigma-Aldrich) to induce heart arrest; pamino-blebbistatin (10 M, Optopharma), a myosin II inhibitor, to suppress a heartbeat without block the action potential; Gadolinium chloride (GdCl3, 0.2 mM and 1 mM, Sigma-Aldrich), a stretch-activated calcium channel inhibitor, to check the influence of Ca 2+ influx in EdCs of the AVC; Oligomycin A (50 g/mL, Sigma-Aldrich), an inhibitor of mitochondrial ATP synthetase, to reduce the ATP level; 5-BDBD (10 M, Sigma-Aldrich), a P2X4 antagonist, to check the function of P2rx4 in the EdCs; NF023 (10 M, Tocris), a P2X1 antagonist, to check the function of P2rx1 in the EdCs; A-317491 (10 M, Sigma-Aldrich), a P2X3 antagonist, to check the function of P2rx3 in the EdCs; A-438079 (10 M, Sigma-Aldrich), a P2X7 antagonist, to check the function of P2rx7 in the EdCs; PPADS tetrasodium salt (20 M, Tocris), a P2X1, X2, X3 and X5 non-selective antagonist, to check the function of P2X1_2_3_5 in the EdCs; BPTU (25 M, Tocris), a P2Y1 antagonist, to check the function of P2Y1 in the EdCs; AR-C118925XX (10 M, Tocris), a P2Y2 antagonist, to check the function of P2Y2 in the EdCs; FK506 (2 M, Abcam), a calcineurin inhibitor, to validate Nfat reporter activity; Janex-1 (0.4 mM, Abcam), a Jak3 inhibitor, to validate Nfat tyrosine phosphorylation (78); Ki8751 (1 M, Tocris), a VEGFR2 inhibitor, to check the effect of Vegfr2 signaling in the EdCs. As a control, the embryos were incubated in E3 solution containing DMSO.Microsurgery to retain the bead into cardiac lumenZebrafish embryos were kept ventral side to the top and embedded with 1% low-melting agarose. After mounted, beads (PureCube Glutathione MagBeads, Cube Biotech) were deposited on top of the agarose with E3 medium. A hole was created in the center of the yolk by forceps and the bead of roughly 30-40 m diameter was inserted into a hole. The bead was pushed anteriorly by the tip of forceps. Once the bead reached the venous pole, suction caused by the heart contraction will move the bead into the cardiac lumen(79).

MagBeads, Cube Biotech) ( 80 ,

 80 81) grafted into embryos. Embryos were kept on their lateral side and embedded into 1% low-melting agarose and the heartbeat was arrested by addition of BDM or tricaine into E3 medium. The magnetic probe was built from three rod-shaped, strong neodymium magnets (diameter, 4 mm; height, 10 mm; S-04-10-AN; Supermagnet) prolonged by a sharpened steel piece with a tip radius of ∼50 m to create a magnetic gradient. The surface of the steel tip was electro-coated with gold to prevent oxidization. The magnetic tweezers were mounted on an inverted epifluorescent microscope (TI-Eclipse, Nikon) combined with a CMOS camera (Hamamatsu), and its position was controlled by a micromanipulator (Injectman 4, Eppendorf). Embryos were filmed with a 20x dry objective (Apo, NA 0.75, Nikon) and a 1.5x magnifier. The microscope was operated with Micro-Manager (Open Imaging). The embryos with the injected beads in the heart were rotated to become aligned with the principal axis of the magnetic tweezer and time-lapse movies were acquired with a time interval of 5 sec. In each experiment, the magnetic force was controlled by the magnet position to shear and/or compress the cells. The positions of the magnet tip and the agarose magbead were recorded in DIC, and the endocardial cells and Ca 2+ signaling were recorded in fluorescence.The magnetic force was calibrated by first charactering the large-scale magnetic force field created by the magnet tip. For this, 2.8 m mono-dispersed magnetic beads were pulled in a viscous test fluid (80% glycerol, viscosity 8.0*10 -2 Pa*sec at 22°C) by the magnet to track the beads velocity as a function of the distance to the magnet, yielding: 𝑉 = 195.4 * 𝑒 -𝑥 31.3 + 31.94 * 𝑒 -𝑥 84.05 𝑉 300 = 0.9134 µ𝑚/𝑠

  C3 probe for overnight at 40 o C. Embryos were washed and amplified the hybridization following the workflow in ACDbio. Embryos were and stored in PBBT at 4 o C prior to confocal imaging. Data analysis Data was analyzed using GraphPad Prism 7 (GraphPad Software). All columns are indicated as mean ± s.d. Statistical significance of multiple groups was determined by one-way ANOVA with Tukey's tests. Statistical significance among two groups was determined by Student's t-test with a two-tailed distribution. Data were considered statistically significant if the p-value <0.05 (*), <0.01 (**), <0.0001 (****).

Acknowledgements

We thank the member's in J.V.'s lab and in N. Mochizuki's lab for discussion and comments on the manuscript; the entire staff of the imaging facility and the fish facility in IGBMC for their assistance; Y.Y.F. in Y.C.H. lab for tabulation of the simulation results; M.S., A.C. and T.B. in N. Mochizuki's lab for their assistance. Funding: J.V. has received funding from the European Research Council (ERC) under the European Union's Horizon 2020 research and innovation program: H.F. was supported from the University of Strasbourg Institute for Advanced Study (USIAS-2017-097), Takeda Medical Research Foundation, Uehara Memorial Foundation, and Cell Science Research Foundation. Author contributions: H.F. and J.V. conceived the project. H.F. designed the work and performed experiments. R.W.C. performed the analysis of Ca 2+ signaling normalization. H.F., J.X. and N. Minc performed the magnetic tweezer experiments. V.V. and Y.C.H. performed the analysis of 3D simulation. N. Minc and N. Mochizuki provided reagents and suggestions. H.F. and J.V. wrote the manuscript. All authors interpreted the data, contributed to data analysis and discussed the manuscript. Competing interests:

The authors declare no competing interests. Data and materials availability: All images

view and anterior to the top. Movie S4. 3D simulation video of wall shear stress in the bead grafted atrial heart region with dynamic wall motion for one cardiac cycle. Shear force intensity was shown by the heat map color.

Movie S5. Single scanned live imaging video (10 msec / frame) of Ca 2+ influx in EdCs (heat map color, fli1a:galff,uas:GCaMP7a) of the bead grafted embryo at 54 hpf. The bead was retained in the atrium (yellow cycle). Arrowheads depict the activated GCaMP7a signal in EdCs. The video was taken ventral view using a lightsheet microscope, anterior to the top. Movie S6. Single scanned live imaging video (10 msec / frame) of Ca 2+ influx in EdCs (heat map color, fli1a:galff,uas:GCaMP7a) of the bead grafted embryo at 54 hpf. The bead was retained in the ventricle (yellow circle). Arrowheads indicate the activated GCaMP7a signal in EdCs. The video was taken ventral view using a lightsheet microscope, anterior to the top. Movie S7. Single scanned live imaging video (5 sec / frame) of Ca 2+ influx in EdCs (green, fli1a:galff,uas:GCaMP7a) and nuclear-localised mCherry signal in EdCs (magenta, kdrl:Nls-mCherry) of the magnetic bead grafted embryo at 2 dpf, and the right panel is the DIC image. The magnetic bead (64 m) was retained in the atrium and the magnetic tweezer was applied after arresting heartbeat. The video was taken left view using an inverted epifluorescent microscope, anterior to the top. White arrowheads depict the initiation of the activated GCaMP7a signal in EdCs.

Movie

Corresponding authors

Materials and Methods:

Zebrafish (Danio rerio) strains, transgenic lines, and mutant lines

The experiments using zebrafish were approved by the animal experimentation committee of the institutional review board of the IGBMC (reference numbers MIN APAFIS#4669-2016032411093030 v4 and MIN 4669-2016032411093030 v4-detail of entry 1) and performed according to the guidelines of the Institute. Embryos were raised in E3 medium and incubated at 28.5 o C. Developmental staging was referred to chronological and morphological criteria [START_REF] Kimmel | [END_REF]. We used the AB strain as wild-type. The following zebrafish transgenic lines were used for experiments: Tg(fli1a:gal4ff) (69), Tg(uas:GCaMP7a) (70), Tg(kdrl:nls-mCherry) ncv5 (71), Tg(fli1a:lifeact-mCherry) ncv7 (72), Tg(tp1:d2EGFP) (73), Tg(fli1a:myr-mCherry) ncv1 (74), Tg(fli1a:h2b-mCherry) ncv31 (24), Tg(myl7:nls-mCherry) ncv11 (75), Tg(uas:RFP) [START_REF] Asakawa | Proc. Natl. Acad. Sci. U. S. A[END_REF], and TgBAC(nfatc1:gal4ff) (65). The Tg(4xnfbr:d2EGFP) ncv531 , TgBAC(twist1b:GFP) ncv534 , Tg(fli1a:GFP-nfatc1) ncv532 , and Tg(uas:VIVIT-mCherry) ncv533 were generated as described in the experimental procedures. We used FK506, a calcineurin inhibitor, which is blocking valve morphogenesis to test the specificity in the Tg(fli1a:lifeact-GFP);Tg(kdrl:nls-mCherry) embryos (25, 40) (Fig. S2D). Such treatment significantly suppressed the reporter expression of Tg(4xnfbr:d2EGFP) embryos (Fig. S10, A andB). In addition, VIVIT peptide overexpression using a selective inhibitor of Nfat dephosphorylation (77), using the TgBAC(nfatc1:galff) driver which is specific to endocardial cells (65)

Genotyping primers

The following genotyping PCR primers were used for amplification: piezo1 (5'-CTATTCCTTTGAGAGCCCTTTG-3' and 5'-GCATGTGGATACAGCAATGAC-3',

Image acquisition and definition of the AVC

To clearly obtain images of embryos, a 0. to make a pTol2-4xnfbr vector. The sequence of 4x tandem repeats was confirmed by DNA sequence. pTol2-4xnfbr vector was inserted with E1b minimal promoter and d2EGFP-polyA sequence to construct the pTol2-4xNfbr-E1b-d2EGFP-pA plasmid.

To monitor the nfat signal inhibition, we established a transgenic line expressing VIVIT-mCherry under the control of Gal4-UAS system; Tg(uas:VIVIT-mCherry) ncv533 . We used a pmCherry-N1 vector as a template and VIVIT peptide (MAGPHPVIVITGPHEE) oligo sequence is inserted upstream of mCherry site by inverse PCR using the following primers: VIVIT-mCherry-S; 5'-atggcgggaccacacccagtaatcgtaattaccggtccacacgaggaggggccccctgtcgctagcATGGTGAGC AAGGGCGAGGAGGATAAC-3' and VIVIT-mCherry-AS; 5'-GGTGGCGACCGGTGGATCCCGGGCCCGCGGTAC-3' to make VIVIT-mCherry-EGFP-hNfatc1 was inserted into pTol2-fli1epEGFP-DEST (Addgene #73491) backbone vector to establish the pTol2-fli1a promoter-GFP-Nfatc1.

Tol2-mediated zebrafish transgenesis was performed by injecting 30 pg of transgene plasmid together with 50 pg of tol2 transposase mRNA, followed by subsequent screening of F1 founders and establishment of stable transgenic strains through selection in F3 generations.

Ca 2+ influx normalisation

To monitor the Ca 2+ signal during heart development, we crossed the Tg(fli1a:gal4ff);Tg(uas:GCaMP7a) line, where a fluorescent Ca 2+ sensor protein GCaMP7a is driven by endocardial gal4, with the Tg(kdrl:nls-mCherry) line, where kdrlpromoter drives nls-mCherry and thus labels endocardial nuclei. To measure the normalised calcium signal of a particular cell over time, we developed software in Matlab (Mathworks) to do the following: For each embryo, the cells imaged move in and out of the focus plane during heartbeat, causing fluctuations in the detectable calcium signal.

Our first step was thus to transform our XYT movies into XYtR movies, where T is the timepoints acquired, t is the timepoint along the cardiac cycle, and R is the iteration of cardiac cycles. To do so, we must account for the changes in heartrate over the period of imaging. Using Matlab, we selected nine region of interests (ROIs) one tenth of the size of the whole image and measured the fluorescence intensity of the nuclear channel over time, Inuclear (T). We calculate the position of the intensity peaks based using ct_getpeaks (86) and select two ROIs where the fluctuations in nuclear channel intensity due to heartbeat are the clearest. Next, we averaged the peak positions acquired from the two ROIs iteratively. Each of these averaged peak positions are assumed to correlate to a specific position within the cardiac cycle. We then calculated the average number of timepoints between these peaks to get the average period of heartbeat tmax. We cut the XYT movie into sections with length of (tmax + 7 images); each centered around the image corresponding to the averaged peak position. We then recombined the sections into a XYtR movie.

For each cell of interest within the embryo, we created XYR movie: The XYtR movie is opened in FIJI, the timepoint along the cardiac cycle that best captures the cell of interest was manually identified, and an XYR movie was created for that timepoint.

Image of average signal: Importing the XYR movie back into Matlab, an image that showed the average calcium and average nuclear signal over the entire course of imaging was calculated. For both channels, each pixel in the final image is the sum of the signal intensity divided over the total number of images, Rmax. Normalised calcium signal: Using the image of the average signal as a guide, we manually drew around the cell of interest to generate the ROI. The normalised calcium signal for the ROI was then calculated using: where Icalcium is the fluorescence intensity of the calcium signal, Inuclear is the fluorescence intensity of the nuclear signal, and the baseline is the minimum Icalcium/Inuclear over the entire XYR movie. Calculate peak position: The position of the peaks was calculated using ct_pulseanalysis (86) with the SMOOTH parameter set to 5.

Whole-mount in situ hybridization (WISH)

The antisense klf2a, klf4a, egr1, p2rx1, p2rx4a and p2rx7 RNA probes labeled with digoxigenin (DIG) were prepared by using an RNA labeling kit (Roche). WISH was performed as previously described (87). Colorimetric reaction was carried out using BM purple (Roche) as the substrate. To stop the reaction, embryos were washed with PBS-T,


Mouad Essani
email: mouad.essani@cea.fr.

Victor Krawiec

Emmanuelle Brackx

Emmanuel Excoffier

Philippe Jonnard

A simple

Keywords: Electron probe microanalysis, Electron microscopy, Thin samples, Microparticles, X-ray emission, Thickness measurement

teaching and research institutions in France or abroad, or from public or private research centers.

INTRODUCTION

Morphological and elementary analysis of thin samples using scanning electron microscopy (SEM) and electron probe microanalysis (EPMA) presents a considerable challenge. The difficulties with regards to analysis with SEM are mainly associated with the lack of knowledge of the sample size in the direction of the incident electron beam (z direction) as imaging is only performed in the x-y plane. The determination of the thickness of samples is important during image analysis and can provide useful information about their 3D geometry.

In EPMA, having the possibility to measure the thickness of thin samples could give insight into the amount of corrections needed to account for absorption and fluorescence effects inside the sample [START_REF] Armstrong | Quantitative elemental analysis of individual microparticles with electron beam instruments[END_REF]. Several approaches for thickness measurement using EPMA were developed for thin films and stratified samples deposited on a substrate [START_REF] Sweeney | Electron Probe Measurements of Evaporated Metal Films[END_REF]; [START_REF] August | A method for determining the mass thickness of thin films using electron probe microanalysis: Determination of mass thickness using EPMA[END_REF]; (Hombourger et al., 2002); (Hombourger et al., 2003); [START_REF] Pereyra | Standardless determination of nanometric thicknesses in stratified samples by electron probe microanalysis[END_REF]. Although these approaches provide satisfactory results, they were tested on thin films with a thickness ranging from few to hundreds of nanometers and their adaptability to micro sized samples is not certain. In addition, most of the available thin film programs [START_REF] Guillaume | Quantitative electron probe microanalysis of multi-layer structures[END_REF]; [START_REF] Llovet | Electron Probe Microanalysis of Thin Films and Multilayers Using the Computer Program XFILM[END_REF]; (Oxford Instruments, n.d.); [START_REF] Pouchou | Surface film X-ray microanalysis: Surface film x-ray microanalysis[END_REF] are not free.

Among other methods that were reported as efficient for thickness measurements are those based on the use of transmission electron microscopy (TEM) such as energy loss spectroscopy (EELS) [START_REF] Zhang | Local thickness measurement through scattering contrast and electron energy-loss spectroscopy[END_REF]; [START_REF] Shi | Thickness measurement of hydrated and dehydrated cryosections by EELS[END_REF] and convergent-beam electron diffraction (CBED) (Delillea, 2001). EELS was shown to provide thickness calculation for samples in a much wider thickness range (~15-500 nm) than the one provided by CBED (~150-300 nm) (Iakoubovskii et al., 2018); [START_REF] Mitchell | Determination of mean free path for energy loss and surface oxide film thickness using convergent beam electron diffraction and thickness mapping: a case study using Si and P91 steel[END_REF]; [START_REF] Egerton | Measurement of local thickness by electron energyloss spectroscopy[END_REF]. In EELS, the thickness can be determined from the low-loss spectrum by means of the Kramers-Kronig (KK) sum-rule [START_REF] Egerton | Measurement of local thickness by electron energyloss spectroscopy[END_REF] or the log-ratio method [START_REF] Malis | EELS log-ratio technique for specimenthickness measurement in the TEM[END_REF]. Despite the fact that thickness calculation with EELS is fast to perform, the accuracy of KK and logratio models depends on the knowledge of the optical refractive index for the former and the inelastic mean free path for the latter. These parameters are however difficult to evaluate for many materials (Iakoubovskii et al., 2018). Moreover, the application of TEM based techniques is limited by both complexity with regards to sample preparation and the sensitivity of image analysis to the thickness of the studied specimen.

An approach for thickness measurement that is adapted to samples thicker than those analyzed with TEM can be advantageous for morphological analysis using electron microscopy.

Typical example of analyses requiring the use of such approaches are those performed on industrial powder composed of a large number of particles exhibiting different sizes. Contrary to TEM, samples can be easily imaged in SEM. This makes the combined use of SEM and EPMA more optimal for morphological and elementary analysis of powder. However, industrial powders, such as those studied in environmental science or in the nuclear industry, contain particles with complex geometry. The thickness of these particles in the direction of the incident electron beam cannot be determined accurately through 2D observations with SEM. The inability to measure the thickness can also be problematic for chemical analysis with EPMA where the correction of size effects is required (Essani et al., 2020a(Essani et al., , 2020b)).

Many attempts were made to perform 3D imaging with SEM [START_REF] Tafti | Recent advances in 3D SEM surface reconstruction[END_REF]. The fastest approaches are those based on the recombination of 2D images obtained from different tilt angles and a further 3D reconstruction (Monteiro et al., 2020). Although good 3D surface reconstruction can be obtained with these approaches, the determination of the sample's thickness through a 3D reconstruction of its volume is not guaranteed. Most advanced procedures for 3D volume representation include serial sectioning using a focused ion beam combined with SEM for imaging (Groeber et al., 2006); (Titze & Genoud, 2016). These methods can be highly efficient with regards to the determination of the material 3D complex microstructure but they are destructive, time consuming and not optimal for tasks as simple as thickness measurements. An optimal morphological analysis of samples with SEM therefore raises the need for a fast and accurate approach for thickness measurements.

We present in this work a fast and simple method that can be used for thickness measurements using an energy dispersive spectrometer (EDS) attached to a scanning electron microscope. Thickness measurements were carried out on NIST (National Institute of Standards and Technology) K411 glass spherical particles and cylindrical particles of U-Ce Oxide. The thickness of each particle was determined from the measured X-ray intensities corresponding to the Kα line of the substrate on which the particle is deposited. Analyses were performed on particles exhibiting different thicknesses, which enabled to assess the validity of the approach on a sufficiently wide thickness range (~ 0.2 to 4 µm).

MATERIALS AND METHODS

Measurements. X-ray measurements were performed using an Oxford EDS attached to a Merlin field-emission SEM. The spectrometer is composed of a silicon drift detector and an AP3 Moxtek window supported by a rigid silicon grid. The take-off angle of the detector is equal to 35°. The working distance and the distance between the spectrometer and the sample were set at 11.5 mm and 19.5 mm, respectively. This enabled to collect a maximum number of emitted photons. In order to obtain a good resolution (~ 125 eV FWHM at Mn Kα) and a counting rate that do not exceed 30% in dead time, all measurements were performed using the longest shaping time and an electron probe current of 0.2 nA. The acquisition time for all the spectra was set equal to 100 s. Uncertainties associated with counting statistics were evaluated by acquiring 8-10 spectra of pure samples (e.g. carbon and silicon) and were estimated within 1-3% for characteristic lines and around 4 to 8% for the background intensities.

Thickness measurements. The thicknesses were determined from the X-ray intensity associated to the Kα line of the substrate on which particles were deposited. This was achieved by probing each particle using a focused electron beam with an energy high enough for the transmission of electrons to occur. After being transmitted through the particle, electrons strike the substrate at a sufficient energy and induce the emission of characteristic X-rays (Figure 1). Being proportional to the number and energy of transmitted electrons, the X-ray intensity emitted from the substrate was used to determine the energy loss of electrons, which enabled measuring the thickness of the analyzed particle. The approach presented in this study is based on the following assumptions: (i) the particle is dense enough and the distance travelled by electrons inside the particle is representative of its thickness, (ii) the composition of the particle is homogeneous and different from the one of the substrate, (iii) the X-ray emission of the substrate is induced by a monoenergetic electron beam at the exit of the particle, (iiii) the electron beam is focused enough so that the influence of its size on the trajectories of electrons inside the particle can be neglected.

Based on the Kanaya-Okayama model (Kanaya & Okayama, 1972), the energy 𝐸 0 ′ (keV) of transmitted electrons can be expressed as:

𝐸 0 ′ = 𝐸 0 (1 - 𝑡 𝑅 ) 3/5 (1)
where 𝐸 0 (keV) is the energy of the primary electrons, 𝑡(µm) is the mean distance travelled by electrons before being transmitted and 𝑅(µm) is the Kanaya-Okayama electron depth range (Kanaya & Okayama, 1972). Equation ( 1) can be re-written as:

𝐸 0 ′ = 𝐸 0 (1 - 𝐽 𝐸 0 1.67 ) 3/5
(2)

with 𝐽 = 𝑡 𝑍 0.89 𝜌 0.0276 𝐴 (3)
where 𝑍 is the mean atomic number of the sample, 𝜌(g cm-3) its density and 𝐴 its mean atomic mass (g).

The approach adopted in this work is based on the determination of the parameter 𝐽 and a further calculation of the thickness. The X-ray spectra of each particle are first measured at different acceleration voltages. This enables to collect characteristic X-ray intensities 𝐼 𝑠 𝑘, 𝑘=1,2..n emitted from the substrate at different electron energies 𝐸 0𝑘 . On the other hand, the dependence of the characteristic intensity 𝐼 on the overvoltage 𝑈 = 𝐸 0 /𝐸 (𝐸 being the ionization energy of the electron shell involved in the production of the characteristic X-rays) can be expressed by (Green & Cosslett, 1961):

𝐼 ∝ 𝑈 𝑙𝑛 𝑈 -𝑈 + 1 (4)
Now if we consider the absorption of X-rays emitted from the substrate inside both the particle and the substrate, we can write for 𝑘 measured intensities 𝐼 𝑠 𝑘 :

𝐼 𝑠 𝑘 ∝ 𝑓 𝑘 (𝜒 𝑠 , 𝐸 0𝑘 ′ )𝑓 𝑘 (𝜒 𝑝 , 𝐸 0𝑘 ) 𝐸 0𝑘 ′ 𝑙𝑛 𝐸 0𝑘 ′ 𝐸 𝑠 -𝐸 0𝑘 ′ + 𝐸 𝑠 𝐸 𝑠 (5)
where 𝑓(𝜒) is the absorption factor, 𝜒 = ( In order to cope with these difficulties, the dependence of the substrate emission on instrumental parameters and absorption factors was canceled by normalizing on the intensity value corresponding to one of the energies 𝐸 0𝑘, 𝑘=1,2..n . Obviously, this suggests that the dependence of the absorption factors 𝑓(𝜒 𝑠,𝑝 ) on 𝐸 0 is no longer considered. However, such a consideration can lead to inaccurate results if the difference between the energies 𝐸 0𝑘 and the one used for normalization is large. 𝐼 𝑠 𝑘 were therefore measured using close energy values can now be expressed as:

( |𝐸 0 𝑘±1 -𝐸 0𝑘 | = 1 keV) so
( 𝐸 0𝑘 𝑙𝑛 ( 𝐸 0𝑘 𝜎 𝑘 𝐸 𝑠 ) -𝐸 0𝑘 + 𝐸 𝑠 𝜎 𝑘 ) ( 𝐸 0 𝑛 2 𝑙𝑛 ( 𝐸 0 𝑛 2 𝜎𝑛 2 𝐸 𝑠 ) -𝐸 0 𝑛 2 + 𝐸 𝑠 𝜎𝑛 2 ) (6)
with

𝜎 𝑖 = (1 - 𝐽 𝐸 0𝑖 1.67 ) 3 5 (7)
According to Green and Cosslett, equation (4) can also be expressed as 𝐼 ∝ 0.365 (𝑈 -1) 1.67 with an error less than 10% in the overvoltage range 1.5 < 𝑈 < 16.

After introducing this formula in equation ( 5), we can express 𝐼 𝑛 𝑘 as:

𝐼 𝑛 𝑘 = ( 𝜎 𝑘 𝐸 0𝑘 -𝐸 𝑠 𝜎𝑛 2 𝐸 0 𝑛 2 -𝐸 𝑠 ) 1.67 (8)
The parameter 𝐽 can afterward be obtained by fitting 𝐼 𝑛 𝑘 values with equations ( 6) or (8). For comparison, the fit was performed in this work with both equations using the Levenberg-Marquardt non-linear least square method (the free GNUPLOT software can be used to that end or the C library lmfit (Wuttke, n.d.) for programming purposes). To a first approximation, the thickness can be considered equal to the mean distance 𝑡 that electrons travel inside the particle. This approximation can be refined by increasing the beam electron energy so as to reduce the amount of side scattered electrons and obtain electron trajectory segments that are more parallel to the direction of the incident electron beam. The thickness was further calculated from the parameter 𝐽 using equation ( 3).

RESULTS AND DISCUSSION

Spherical K411 glass microparticles. Analyses were performed on five spherical particles of K411 glass, two deposited on a nickel (Ni) substrate (particles S1 and S2) and three on aluminum (Al) (S3, S4 and S5) (Figure 2). The X-ray spectra of all the particles showed an intense characteristic X-ray line of the substrate indicating the significant transmission of electrons through the particles. Because the energy of transmitted electrons 𝐸 0 ′ increases as the thickness decreases, the substrate emission was observed to be more important for particles with low diameters (e.g. X-ray spectrum of particles S1 and S2 shown in Figure 3).

Particles were probed at a normal incidence of the electron beam using five to seven electron energies 𝐸 0𝑘 . This enabled collecting different X-ray intensities of the substrate. The normalized intensities 𝐼 𝑛 were further determined for each energy and fitted using equation ( 6) and ( 8). The parameter 𝐽 was obtained through the fitting, which enabled calculating the thickness of each particle using equation ( 3). An example is shown in Figure 4 where the normalized intensities obtained by analyzing particles S1 and S2 are presented along with their fitting.

The thicknesses were calculated for all the particles from 𝐽 values using both equations ( 6) and ( 8) (table 1). The results presented in table 1 indicate clearly the good accuracy of the approach with regards to thickness measurements. The deviations between the diameters of particles and the mean values of their calculated thicknesses are less than 10%. Results obtained with equations ( 6) and ( 8) also show good agreements with deviations between calculated mean thicknesses not exceeding 11%. Although this shows the validity of both equations, with the except of particle S5, equation ( 6) generally provides more accurate thickness calculations. This suggests that equation (4), adopted in equation ( 6), probably gives a better description of the dependence of the characteristic X-ray intensity on the overvoltage.

It is important to note that thickness measurements were performed using raw characteristic intensities of the substrate, i.e. the background was not extracted from the measured spectra.

Although it is more practical to work with net intensities (𝐼 𝑠 -𝐵 𝑠 , 𝐵 𝑠 being the Bremsstrahlung intensity of the substrate) the background associated to the substrate is hard to evaluate. This is due to the contribution of the particle's Bremsstrahlung to the measured background. The measured characteristic X-ray intensities of the substrate should therefore be intense enough so that deviations between net intensities and raw ones can be neglected. In this work, good measurements were obtained when the condition 𝐵 𝐼 𝑠 ≤ 5% was satisfied, with 𝐵 the measured Bremsstrahlung intensity under the X-ray line of the substrate.

Cylindrical microparticles of U-Ce oxide. Thickness measurements were carried out on three particles of U-Ce oxide (C1, C2 and C3) deposited on an Al substrate. Image analysis with SEM showed particles exhibiting a cylindrical form with a diameter in the interval [0.20 : 0.24 µm] for C1, [0.53 : 0.60 µm] for C2 and [0.80 : 0.88 µm] for C3 (Figure 5). The red circles in Figure 5 indicate the zones of analysis performed on each particle. The thicknesses are therefore determined locally at the region where the electron beam enters the particle.

The characteristic X-ray intensities of the Al substrate were further measured using different electron energies 𝐸 0𝑘 ranging from 12 to 19 keV. After fitting the normalized intensities 𝐼 𝑛 , the parameter 𝐽 was obtained for each particle which enabled calculating their thicknesses.

Table 2 shows the calculated thicknesses corresponding to the regions presented by red circles in Figure 5. It can be seen that both equations provide results that are in satisfactory agreements with the expected values. Less accuracy is however obtained in the case of particle C2-zone 1 where the calculated thicknesses are slightly overestimated. This being said, some U-Ce oxide particles do not have a perfectly cylindrical shape and their thicknesses might differ, to a lesser extent, from their widths. Another parameter that could also contribute to the observed deviation is the influence of electron energies on thickness measurements.

As shown in table 3, thicknesses determined for C2-zone 1 depend on the values of energies 𝐸 0𝑘 . It can be observed that calculated thicknesses are lower at high energies. A probable cause of such an observation can be related to the decrease of elastic scattering of electrons inside the particle as 𝐸 0 increases. In fact, as the electron energy decreases, the amount of scattered electrons becomes important. Electrons might therefore undergo path lengths larger than the diameter of the cylindrical particle leading to an increase of their energy loss. The calculated thicknesses are therefore expected to be somewhat higher when working with low energies. On the other hand, the deviation between results obtained with equations ( 6) and ( 8) is significant at high energies (~30%). It is likely that this results from errors associated to the approximation of equation (4) as a power of (𝑈 -1).

CONCLUSION

Simple thickness measurements using EPMA is achieved. The proposed approach was validated on particles with a size smaller than the electron depth range. Thicknesses were determined by means of the substrate X-ray emission on which the particles were deposited.

The X-ray intensity emitted from the substrate can provide a good estimation of the energy loss of electrons inside the particle. Being proportional to the mean distance travelled by electrons inside the particle, the energy loss of electrons enables the determination of the thickness.

The use of normalized X-ray intensities emitted from the substrate enables, to a reasonable degree, taking account of the intensity loss caused by absorption effects and instrumental parameters. This allows obtaining a direct dependence between measured intensities of the substrate and the energy loss of electrons inside the particle. The thickness can be further determined by fitting the normalized intensities using equations ( 6) and ( 8). Although equation ( 8) might lead to inaccurate thickness calculation at high electron energies, it is recommended to use both equations in order to verify the consistency of the determinations.

The fast thickness measurements provided by this approach can be advantageous for powder characterization as it offers the possibility of performing optimal morphological analysis of a large number of particles. Thickness mapping of particles or thin samples is an interesting application that could also benefit from the improved timeliness of thickness measurements offered by the approach.

It is worth noting that the necessity to work with K lines of the substrate may prevent the application of the approach to substrates composed of heavy elements (e.g. electron probes operating at a maximum acceleration voltage of 40 kV). The adaptability of the method to L and M lines is therefore an interesting topic and will probably be discussed in future studies.

Other limitations of the method that should also be mentioned are related to i) thickness measurements of nanoparticles where the size of the electron beam becomes an influencing parameter and ii) analysis of hollow or highly porous particles in which the thickness does not correspond to the distance travelled by electrons in matter.

  coefficient and 𝜃 the take-off angle of the X-ray detector. The indices 𝑠 and 𝑝 stand for absorption inside the substrate and the particle, respectively. 𝐸 𝑠 (keV) is the photon energy corresponding to the K line of the substrate element.It is worth noting that the X-rays generated inside the substrate undergo different path lengths before reaching the detector. Absorption factors 𝑓(𝜒 𝑠,𝑝 ) inside both the substrate and the particle are therefore hard to evaluate. Moreover, determining the energy loss of electrons from equation (5) requires accounting for the intensity loss caused by absorption in the X-ray detector. Determining the detector efficiency adds more complications, especially in cases where the thickness of layers in the spectrometer window are not known with sufficient accuracy.

  Figure captions

Figure

  Figure 2. SEM Images of K411 glass spherical microparticles showing a diameter of 0.75

Figure 3 .

 3 Figure 3. X-ray spectra acquired at an electron energy 𝑬 𝟎 = 𝟐𝟎 keV for particles S1 and

Figure 4 .

 4 Figure 4. Normalized intensities 𝑰 𝒏 𝒌 obtained by analyzing particles S1 and S2 and their fit using equations (6) and (8). The values of 𝑰 𝒏 𝒌 were obtained using five electron energies 𝑬 𝟎𝟏 = 18 keV, 𝑬 𝟎𝟐 = 19 keV, 𝑬 𝟎𝟑 = 20 keV, 𝑬 𝟎𝟒 = 21 keV and 𝑬 𝟎𝟓 = 22 keV with 𝑬 𝟎𝟑 the median value used for normalization.

Figure 5 .

 5 Figure 5. SEM images of cylindrical microparticles of U-Ce oxide.

  that the energy difference between the lowest (or highest) accelerating voltage and the normalizing accelerating voltage remains small. The intensity used for normalization was chosen as the one corresponding to the median energy 𝐸 0 𝑛

			. The
			2
	normalized intensities 𝐼 𝑛	𝑘 = 𝐼 𝑠	𝑘 /𝐼 𝑠 𝑛 2

ACKNOWLEDGMENTS

We acknowledge the financial support received from the Cross-Disciplinary Program on Instrumentation and Detection of the CEA, the French Alternative Energies and Atomic Energy Commission.

Conflicts of interest: