Alexandre Khaldi

Daniel Falk

Katarina Bengtsson

Ali Maziz

Daniel Filippini

Edwin Nathaniel D Robinson

W H Jager

Edwin W H Jager

Patterning highly conducting conjugated polymer electrodes for soft and flexible microelectrochemical devices

Keywords: Vapor phase polymerization, Printing, soft Lithography, Conjugated Polymers actuators, patterning, microfabrication

ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

The recent and rapid development of bioelectronic devices such as implantable or wearable electronics, neural interfaces, drug delivery systems, and soft actuators have highlighted the need for soft, flexible, and stretchable electrodes and wires. Conjugated polymers (CPs) are the materials of choice for interfacing electronic devices with natural tissue and cells due to their combination of electronic and ionic conductivity, biocompatibility, low cost, the ease of processing, and their ability to undergo reversible oxidation and reduction reactions. [START_REF] Smela | Conjugated Polymer Actuators for Biomedical Applications[END_REF][START_REF] Gelmi | Direct Mechanical Stimulation of Stem Cells : A Beating Electromechanically Active Scaffold for Cardiac Tissue Engineering[END_REF][START_REF] Stavrinidou | [END_REF][START_REF] Wadhwa | Electrochemically Controlled Release of Dexamethasone from Conducting Polymer Polypyrrole Coated Electrode[END_REF][START_REF] Williamson | Controlling Epileptiform Activity with Organic Electronic Ion Pumps[END_REF][START_REF] Ouyang | Enhanced PEDOT Adhesion on Solid Substrates with[END_REF][START_REF] Hardy | Biomimetic Conducting Polymer-Based Tissue Scaffolds[END_REF] CPs with good chemical stability exhibiting electronic conductivity above 1000 Scm -1 are now common. 8- 10 However, these CP layers are frequently coated without patterning, and must therefore be patterned, for instance, to form individual electrodes, in order to obtain functional devices. [START_REF] Choi | Micro-Patterning of Vapor-Phase Polymerized poly(3,4-Ethylenedioxythiophene) (PEDOT) Using Ink-Jet Printing/soft Lithography[END_REF] A variety of techniques have been used to pattern these materials at the micro/nano scale. [START_REF] Smela | Controlled Folding of Micrometer-Size Structures[END_REF][START_REF] Edberg | Patterning and Conductivity Modulation of Conductive Polymers by UV Light Exposure[END_REF] This can be achieved by electropolymerization onto patterned gold electrodes, but the poor adhesion of the gold layer to both the CP and the underlying substrate limits device lifetime. [START_REF] Smela | Controlled Folding of Micrometer-Size Structures[END_REF][START_REF] Edberg | Patterning and Conductivity Modulation of Conductive Polymers by UV Light Exposure[END_REF][START_REF] Smela | Microfabrication of PPy Microactuators and Other Conjugated Polymer Devices[END_REF][START_REF] Jager | Microrobots for Micrometer-Size Objects in Aqueous Media: Potential Tools for Single-Cell Manipulation[END_REF][START_REF] Jager | Microfabricating Conjugated Polymer Actuators[END_REF] The subtractive patterning of a CP layer by laser ablation [START_REF] Lee | Microstructuring of Polypyrrole by Maskless Direct Femtosecond Laser Ablation[END_REF] or reactive ion etching [START_REF] Khaldi | Conducting Interpenetrating Polymer Network Sized to Fabricate Microactuators[END_REF] is extremely difficult to perform on multi-layered materials, as it is difficult to ensure that only the CP material is removed without disturbing any underlying polymer layers. Over-oxidation (electrochemical or oxidative deactivation) of CP layers can also be used for patterning, but this method only locally disrupts the electrical conductivity, while physical removal of the electrode material is also often required. [START_REF] Tehrani | Patterning Polythiophene Films Using Electrochemical over-Oxidation[END_REF] Rational fabrication of bioelectronics devices based on CPs requires new patterning techniques that are compatible with batch fabrication processes that use bottom-up microfabrication techniques. PEDOT-PSS dispersed in water, e.g. Clevios S TM from Heraeus, is directly patternable by screen printing, [START_REF] Berg | Screen Printed Conductive Pastes for Biomedical Electronics[END_REF] is able to present high electronic conductivities [START_REF] Palumbiny | Molecular Reorientation and Structural Changes in Cosolvent-Treated Highly Conductive PEDOT : PSS Electrodes for Flexible Indium Tin Oxide-Free Organic Electronics[END_REF][START_REF] Palumbiny | The Crystallization of PEDOT : PSS Polymeric Electrodes Probed In Situ during Printing COMMUNICATION[END_REF] but has diminished electrochemical capacity per unit mass due to the mass contribution of the counterions and additives used. In the case of actuator applications, vapor phase polymerization (VPP), in combination with reactive ion etching, has enabled batch fabrication of simple CP actuator devices that can operate in air. [START_REF] Maziz | Top-down Approach for the Direct Synthesis, Patterning, and Operation of Artificial Micromuscles on Flexible Substrates[END_REF][START_REF] Khaldi | Conducting Interpenetrating Polymer Network Sized to Fabricate Microactuators[END_REF][START_REF] Maziz | Demonstrating kHz Frequency Actuation for Conducting Polymer Microactuators[END_REF] CP actuators utilize the volumetric expansion caused by the oxidation or reduction of the CP.

A typical CP actuator structure (see Figure 1) is the so-called trilayer configuration, where the outer layers consist of CPs and the middle layer is a polymer electrolyte (ion reservoir). When a voltage is applied, the CP layer at the cathode is reduced and the CP layer at the anode is oxidized. Cations and/or anions migrate, compensating the charges formed on the CP chains, maintaining electroneutrality, and inducing opposing volumetric changes in the electrodes. The simultaneous expansion and contraction of the CP layers sandwiching the polymer electrolyte causes the trilayer beam to bend. This bending is reversible, so applying an alternating potential causes the beam to oscillate. [START_REF] Vidal | Long-Life Air Working Conducting Semi-IPN/ionic Liquid Based Actuator[END_REF][START_REF] Vidal | Semi-Interpenetrating Polymer Networks : A Versatile Concept for the Design of Optical or Mechanical Electroactive Devices Claude Chevrot a and Dominique Teyssi E[END_REF] These trilayer CP actuators, operable in air, have been scaled down to the micro-domain, and multiple bending beam type microactuators have been fabricated in parallel. [START_REF] Khaldi | Synergetic PEDOT Degradation during a Reactive Ion Etching Process[END_REF] Unfortunately, the previously-reported parallel actuators could not be operated individually as the micropatterning method did not allow individual independent electrical connections. Individually controlled actuation has been demonstrated at the centimeter scale using classically-micropatterned metal electrodes with electrodeposited polypyrrole (PPy). [START_REF] Jager | Patterning and Electrical Interfacing of Individually Controllable Conducting Polymer Microactuators[END_REF] However, the latter method required flipping the substrate during processing, making the alignment of the various layers difficult, hampering microfabrication and hindering scale-up and commercial development. To pattern CP electrodes at microscale resolution, we recently developed a bottom-up microfabrication method; unfortunately, these trilayer actuators did not show the large deformations expected with CP actuators because the electrolyte membranes used had relatively low ionic conductivity. [START_REF] Khaldi | Bottom-up Microfabrication Process for Individually Controlled Conjugated Polymer Actuators[END_REF][START_REF] Khaldi | Flexible Micromanipulators Comprising Polypyrrole Trilayer Microactuators[END_REF] A few attempts have been made to obtain micropatterned CP layers using soft lithography and VPP, but the adhesion of the resulting electrode was insufficient for devices undergoing large deformations. [START_REF] Choi | Micro-Patterning of Vapor-Phase Polymerized poly(3,4-Ethylenedioxythiophene) (PEDOT) Using Ink-Jet Printing/soft Lithography[END_REF][START_REF] Winther-Jensen | Vapor-Phase Polymerization of 3,4-Ethylenedioxythiophene: A Route to Highly Conducting Polymer Surface Layers[END_REF] Here, we present two patterning methods, micro-Contact Printing (µCP) and syringe-based printing, suitable for VPP. Although we focus on CP actuator processing in this paper, these methods are suitable for microfabricating many kinds of devices comprising CPs. These two methods allow the patterning of micro-to millimeter-sized electrodes and wires. The resolution of each patterning technique was evaluated by printing circular or square micrometer-sized shapes for µCP and millimeter-sized lines for syringe-based printing. The resolution achieved with both techniques enabled us to demonstrate active CP electrodes connected to contacts with thin addressing lines. Finally, the capabilities of the novel patterning methods are demonstrated by fabricating a soft actuator based on CP technology.

Results and discussion

Polymer electrolyte

We have chosen to use the semi-interpenetrating network (IPN) poly(ethylene oxide)nitrilebutadienerubber (PEO-NBR) to obtain a robust solid polymer electrolyte (SPE) with sufficient ionic conductivity. [START_REF] Maziz | Top-down Approach for the Direct Synthesis, Patterning, and Operation of Artificial Micromuscles on Flexible Substrates[END_REF] To fabricate high performance CP actuators, the center polymer electrolyte layer (Figure 1) should be a membrane swollen by an electrolyte with a high ionic conductivity, allowing efficient ion migration between the adjacent CP electrodes, and capable of high bending deformation without fracture. This currently limits the choice to three membrane materials: poly(ethylene oxide)-nitrilebutadienerubber interpenetrating network (PEO-NBR IPN) membrane, [START_REF] Goujon | Flexible Solid Polymer Electrolytes Based on Nitrile Butadiene Rubber / Poly(ethyleneoxide) Interpenetrating Polymer Networks Containing Either LiTFSI or EMITFSI[END_REF][START_REF] Festin | Robust Solid Polymer Electrolyte for Conducting IPN Actuators[END_REF] the commercial porous polyvinylidene fluoride (PVDF) membrane [START_REF] Gaihre | Pushing the Limits for Microactuators Based on Electroactive Polymers[END_REF] and the PVDF ionic liquid gel membrane. [START_REF] Fukushima | Fully Plastic Actuator through Layer-by-Layer Casting with Ionic-Liquid-Based Bucky Gel[END_REF] The porous PVDF membrane cannot be fabricated on a substrate using spin coating. The PVDF ionic liquid gel is difficult to use because common patterning sequences require immersing the membrane in various solutions, leading to leaching of ionic liquids out of the membranes and limiting the robustness of such processes. Hence, the semi-IPN PEO-NBR was chosen.

Vapor-phase polymerized PEDOT

VPP methods have shown to be suitable for obtaining strong adhesion between CP electrodes and the membranes described above. VPP is a heterogeneous oxidative polymerization reaction between an oxidative surface and a gaseous monomer. VPP was carried out for 30 min at 40 C under a vacuum atmosphere of 1 mPa (Figure 2a).

We have previously used VPP to fabricate CP trilayer actuators with an interpenetrating polymer network based on PEO-NBR as the center electrolyte layer. [START_REF] Goujon | Flexible Solid Polymer Electrolytes Based on Nitrile Butadiene Rubber / Poly(ethyleneoxide) Interpenetrating Polymer Networks Containing Either LiTFSI or EMITFSI[END_REF][START_REF] Festin | Robust Solid Polymer Electrolyte for Conducting IPN Actuators[END_REF] The process started with VPP of PEDOT on the substrate, followed by the PEO-NBR deposition and polymerization, and finished with a second VPP of PEDOT on top of the membrane. [START_REF] Khaldi | Fabrication and Adhesion of Conjugated Polymer Trilayer Structures for Soft, Flexible Micromanipulators[END_REF] The structure was cured for 1 hour at 80°C to obtain crosslinking at the interfaces (described below). [START_REF] Maziz | Top-down Approach for the Direct Synthesis, Patterning, and Operation of Artificial Micromuscles on Flexible Substrates[END_REF]

Effect of PEGM-PEGDM on polymerization

Oxidant solutions were mixed to yield between 0 and 50 wt.% ethylene oxide methacrylates (PEGM-PEGDM, see Experimental section) added to 40 wt.% Fe (Tos) 3 oxidant solution (in Ethanol/Butanol mixture). It has been shown that various additives and ethylene oxide derivatives [START_REF] Fabretto | Polymeric Material with Metal-Like Conductivity for Next Generation Organic Electronic Devices[END_REF][START_REF] Winther-Jensen | Vapor-Phase Polymerization of 3,4-Ethylenedioxythiophene: A Route to Highly Conducting Polymer Surface Layers[END_REF][START_REF] Bubnova | Semi-Metallic Polymers[END_REF] help to reduce the crystallization of Fe and reduce the rate of polymerization, leading to highly ordered CP films with high electronic conductivity (on the order of 10 3 Scm - substrates with the addition of a post-processing step that induces the formation of covalent bonds between the layers. [START_REF] Maziz | Top-down Approach for the Direct Synthesis, Patterning, and Operation of Artificial Micromuscles on Flexible Substrates[END_REF] Figure 2b reports the thicknesses and the conductivity of the CP layers resulting from VPP with solutions with varying PEGM-PEGDM content. VPP carried out with an oxidant solution without the ethylene oxide methacrylates leads to a thickness of 100 nm.

The highest measured conductivity was 800 Scm -1 for a film of 200 nm thickness prepared from an oxidant solution containing 30 wt% PEGM-PEGDM. The high conductivity of this CP layer allows (metal-free) electropolymerization of subsequent polymer layers. [START_REF] Khaldi | Fabrication and Adhesion of Conjugated Polymer Trilayer Structures for Soft, Flexible Micromanipulators[END_REF][START_REF] Temmer | In Search of Better Electroactive Polymer Actuator Materials: PPy versus PEDOT versus PEDOT-PPy Composites[END_REF]

Effect of PEGM-PEGDM on surface interaction

Interactions between the oxidant solution and several substrates used in our patterning techniques have been analyzed. The contact angle (ϴ) of the oxidant solution with varying PEGM-PEGDM content on different substrates is presented in Figure 3. We measured the contact angle of the oxidant solutions on PDMS with and without O 2 plasma treatment, glass, polyvinyl alcohol (PVA), PEO-NBR IPN, and PVDF. The contact angle of the oxidant solution for each of the formulations was very high (above 50 °) on PDMS, glass, and PEO-NBR substrates. When the PDMS was treated with an O 2 plasma, the observed contact angle decreased significantly. The oxidant solution had a stronger interaction with the PVDF and PVA substrates, but the contact angle surprisingly increased to 38 ° when we add 10 wt.% PEGM-PEGDM to the oxidant solution. The value of the contact angle on the PVDF and PVA decreased again with increasing PEGM-PEGDM content. The tunable affinity between the oxidant solution and substrates has consequences on the quality of the reproduction of patterns after polymerization, as described in the coming sections.

Oxidant patterning

To achieve highly conductive high-resolution patterns of CP electrodes, we first pattern the oxidant solution and then perform VPP, instead of directly printing or patterning a CP layer. As mentioned previously, two different techniques have been developed to pattern the oxidant solution: µCP and syringe-based printing. For µCP, square patterns were created on a PDMS stamp and used to verify the technique's ability to reproduce shapes with sharp corners. The µCP process, and syringe-based printing are illustrated in Figure 4. After patterning the oxidant with one of these techniques, VPP is performed on the oxidant, forming a nicely-patterned CP layer. Next, a multi-layered structure comprising a polymer electrolyte and a second patterned CP layer can be formed, which thereafter can be processed using laser ablation (Figure 4.) 2) The PDMS stamp is removed from the substrate and the oxidant solution adheres to the pattern of the PDMS stamp. 3) The oxidant pattern is reproduced onto a substrate covered by either a PVA or a PVDF layer. 4) The stamp is removed, leaving the oxidant pattern deposited onto the substrate. The syringe-based printing must be realized onto a glass slide. The vapor phase polymerization is following the creation of this oxidant pattern. A polymer electrolyte layer can be deposited before the deposition of a subsequent CP layer. The laser ablation is used to cut cleanly the final device.

µ-contact printing

The interaction between the fluid (oxidant solution) and stamp and substrates are particularly important when using µCP, as these interactions influence the resolution and shape of the resulting pattern. To transfer the oxidant solution from a temporary substrate (onto which the oxidant solution was applied via spin coating) to the stamp, the affinity of the oxidant solution for the temporary substrate must be less than its affinity for the stamp. Surface-treating the PDMS stamp with oxygen plasma dramatically increases the solution's affinity for the PDMS. In the second step of the process, the solution's affinity for the target substrate should be even greater than its affinity for the PDMS stamp. The contact angle measurements for all compositions of the oxidant solution show that the solutions affinity for glass is less than that for plasma-treated PDMS, which in turn is less than its affinity for targets substrate of either PVDF or PVA. In other words, the process outlined in figure 3 moves the liquid from substrates on which it has the highest contact angle to materials on which it has progressively lower contact angle ( Glass >  PDMS >  PVA ). The combination of µCP with vapor phase polymerization allowed us to easily create well-defined micro-patterned electrodes with high conductivity. Figures 5a-b show the micro-patterns obtained on a PVA substrate. It shows that it is possible to produce highly defined micro-patterns on such a surface. This technique allows reproduction of different patterns with sizes between 100 and 500 µm and a minimum spacing distance of 50 µm onto both PVA and PVDF substrates (Figure S1). Circular, rather than square, patterns are obtained when 10 wt.% PEGM-PEGDM is present in the oxidant solution, while more accurate replication of the square pattern of the stamp was obtained for 30 wt.% additive content in the oxidant solution onto both PVA and PVDF substrates. This observation is consistent with the maximum in measured contact angle between the oxidant solution and all substrates. This result indicates that that we can fine-tune the shape of the pattern using the formulation of the oxidant solution, which we believe warrants further study.

One advantage of µCP is that a complete layer of complex device architecture can be patterned in a single step. To demonstrate this, we made a PDMS stamp with the actuator lay-out of the first layer comprising two individually controllable electrodes/actuators, leads/wires, and contact pads. We used µCP to fabricate millimetric electrodes with 600 µm wide conducting lines in a single printing step (Figure 5c). The high conductivity obtained for the electrodes allowed us to cover the thin PEDOT electrodes with a PPy layer in a subsequent electropolymerisation step with homogeneous thickness (8 µm, Figure 5c). Unfortunately, we have been unable to use this technique to fabricate CP electrodes on the highly ionically conductive membrane PEO-NBR due to the insufficient affinity between the oxidant solution and the PEO-NBR membrane (ϴ>60°) compared to affinity for the plasma treated PDMS stamp (ϴ<40 °). O 2 plasma treatment of the PEO-NBR membrane reduces the surface energy (measured contact angle), but induces other problems such as penetration of the oxidant solution into the membrane, causing the membrane to swell. If one wants to fabricate patterned multilayered CP device with this method, new SPE membranes need to be developed. The µCP pattern obtained with the same process but with 30 % PEGM-PEGDM, c) Using µCP, large electrodes (≈10 mm 2 ) with contact pads and connecting lines (600 µm wide) were obtained with VPP on an oxidant layer with 10 wt% PEGM-PEGDM in a single printing step. These active electrodes can then be homogeneously covered by a thick layer of PPy using electropolymerisation. Note that the contact pads were not exposed to the electropolymerisation solution and thus are not covered with PPy.

Syringe-based printing

For syringe-based printing, a different interaction between the oxidant solution and the substrate is necessary. To prevent the solution from spreading onto the substrate, but rather retain the shape of the pattern being printed, the affinity of the solution with the substrate should be relatively low (ϴ > 50 °). Both glass and PEO-NBR substrates have a surface energy appropriate for patterning the oxidant layer using syringe-based printing, thus enabling the fabrication of patterned CP trilayer devices.

Figure 6 shows the principle of fabricating individually controlled trilayer actuators with individual contacts on the same polymer film using syringe-based printing. The line resolution of the patterns obtained in this work was ~1 mm. The resolution is limited by the size of the needle, solution viscosity, the oxidant solution interaction between the tip of the needle and the substrate, the gap between needle and substrate, and the spreading of the material on the surface. Further optimization of these printing parameters would allow higher resolution patterns to be printed. The second step is the spin coating and the curing of the PEO-NBR membrane. The third step is the patterning of the second layer of electrodes using the same method used for the first electrode layer. Special attention was paid to align the actuator electrodes. The last step is to peel the trilayer membrane from the glass substrate (not shown). b) A photograph showing the finished device, designed as a finger with two joints that can be individually controlled. It comprises 3 actuators, integrated electrical leads, and contacts. Actuator 1 forms the first joint and can be controlled via the outermost leads. The second joint is formed by actuators 2 and 3 that are electrically interconnected and can be controlled by the inner leads.

Laser ablation

As the final processing step (after polymerization and before removal from the supporting substrate), the actuator device needs to be cut out from the rest of the membrane to release the actuating beam. Previously, this was done using reactive ion etching (RIE) where the final device structure was defined by a photoresist pattern. [START_REF] Khaldi | Synergetic PEDOT Degradation during a Reactive Ion Etching Process[END_REF] This was possible since the continuous CP layer on top of the SPE protected the SPE from swelling caused by the photoresist processing.

However, if the top CP layer, i.e. the layer on top of the SPE, is already patterned, as is the case for our VPP patterned CP electrodes, photoresist cannot be used since parts of the SPE are exposed to the solutions used in the photoresist process, causing swelling. [START_REF] Jager | Conducting Polymers as EAPs: Microfabrication[END_REF] Here, we used laser ablation to define the final device shape. Laser ablation has a lower resolution than photolithography combined with RIE, but is more versatile in rapid prototyping applications. We have patterned an individual beam with 60 x 500 x 2000 µm 3 geometry and a contact pad (Figure 7b), and millimeter-sized hands (Figure 7d) to illustrate the usefulness of this technique for this type of device. After swelling the devices in the ionic liquid 1-Ethyl-3-methylimidazolium bis(trifluoromethylsulfonyl)imide (EMITFSI), we evaluated the actuation of these hands by connecting them at the palm (Video S1). The actuation properties of this milli/micro device are interesting. For the hand, we achieved ~1 mm displacement when stimulating with a ± 2 V square wave potential at 0.1 Hz, which gives a strain of ε=2,2 %. (See experimental section for the definition of strain ε. The active length was 2.5 mm and total actuator thickness 70 μm.) The performance can be further improved by adding an extra electropolymerization step during the process to provide a more electroactive material. [START_REF] Khaldi | Fabrication and Adhesion of Conjugated Polymer Trilayer Structures for Soft, Flexible Micromanipulators[END_REF]

Actuation test

Figure 8a shows an array of free-standing syringe-based printed trilayer actuators (10 mm long, wide and 70 μm thick) following the method illustrated in Figure 6. One of these line actuators was manually cut to investigate the actuation properties (Figure 8b). PPy electrochemical deposition was performed for 2 hours at 0.1 mA cm -2 at -18 °C from an electrolyte solution (0.1M LiTFSI and 0.1M pyrrole in propylene carbonate). [START_REF] Khaldi | Bottom-up Microfabrication Process for Individually Controlled Conjugated Polymer Actuators[END_REF] The fabricated trilayer was immersed into the ionic liquid EMITFSI until absorption (swelling) ceased (~6 hr) and its actuation behavior was characterized by applying a ± 1V square wave potential at 0.025 Hz to the electrodes (Figure 8b). The total thickness of this actuator was ~100 µm. The large bending typical of CP actuators was also observed for these printed actuators. A maximum deflection/bending of 10 mm (ε = 2 %) was achieved similar to previous unpatterned devices, [START_REF] Vidal | Long-Life Air Working Conducting Semi-IPN/ionic Liquid Based Actuator[END_REF] indicating that the patterned actuators perform similarly to their unpatterned counterparts.

Patterning in this manner eliminates many of the negative effects inherent in other techniques, opening a new path for bottom-up fabrication of individually fabricated polymer actuators. To demonstrate the potential of combining the two CP patterning techniques presented with laser ablation, a device containing two individually addressable trilayer actuators has been fabricated (Figure 8c). Two adjacent trilayer actuators from the array of actuators shown in the Figure 8a have been neatly cut from the membrane using laser ablation, leaving a ~9 mm long segment of the polymer electrolyte layer connected at the bottom, thus forming a device with two individually controlled digits. The device, folded in half so that the actuators oppose one another, clearly demonstrates that with further process and design optimization the combination of the developed CP patterning methods with laser ablation enables the development of complex soft microrobotic devices with individually controlled actuators.

Conclusion

We have demonstrated two complementary techniques -µ-contact printing and syringe-based printing -to create highly conducting CP patterns via Vapor Phase Polymerization. This was enabled through a study that yielded an understanding of the tunable wetting of the oxidation solution on the various substrates involved and a subsequent optimization of the process parameters, including the PEGM-PEGDM content. This improved VPP technique achieved a more uniform, higher conductivity film, with tuneable wetting for use on various substrates.

Patterns down to 100 µm can be fabricated with a good resolution using µ-contact printing and VPP. Line widths down to 1 mm can easily be achieved using syringe-based printing and VPP. µCP combined with VPP is useful for fabricating micro-electrochemical devices of any arbitrary shape with high resolution, and especially allows complete layouts to be printed in a single patterning step. Syringe-based printing is very promising for fabricating functional devices at the millimeter and submillimeter scales, especially for multilayered systems. Moreover, syringebased printing has the potential for use with 3-D printing to obtain 3-D electrodes and even encapsulated devices. In these first demonstrations, the resolution was not optimized and can likely be improved by further adjusting the processing parameters such as the stamps patterns and their surface density, the formulation of the oxidant solution, the choice of the substrate and the chemical functionalization of the surface of the stamp. Finally, we showed that it is possible to fabricate fully functional electrochemical devices, in this case trilayer actuators, using the new printing methods. In combination with laser ablation this should enable the fabrication of complex soft micro-tools. The devices fabricated using these newly developed patterning methods function as good as non-patterned devices, demonstrating that the patterning does not result in any significant negative effects, thus opening the way of new bottom-up fabrication of and AIBN (3wt.% of the methacrylic monomers). The mixture of PEO and NBR was then spincoated. The thickness of the polymer films depends on the selected spin coater speed, time and solution viscosity. The substrate was then kept at 80°C for 10 h.

Oxidant solution preparation: The 40% oxidant solution was poured in a vial. The ethylene oxide acrylate oligomers (PEGM-PEGDM) were then added to the solution and stirred with a magnetic bar. The ethylene oxide acrylate oligomers derivatives had a fixed proportion of monomers PEGM/PEGDM of 75/25 for each of the prepared solutions.

Vapor phase polymerization: The oxidant solution was spincoated on the substrate at 1000 rpm for 30 s on a POLOS spin-coater on glass slides. The coated substrates were placed on the hot plate in the vacuum oven (1 mPa) containing a Petri dish with 2 drops of EDOT and maintained at 40 °C for 30 min. Thereafter the substrates were taken out, washed with ethanol and dried at room temperature at 1 mPa for 2 h. Electropolymerisation: The polypyrrole (PPy) layers were electrodeposited in a two-electrode configuration at a constant current of 0.1 mAcm -2 for 2 hours using an Ivium compactstat or IviumStat (Ivium Technologies, Eindhoven, The Netherlands) on the VPP PEDOT layer from a 0.1 M Pyrrole, 0.1 M LiTFSI and 1 wt.% water in a propylene carbonate (PC) solution for 2 hours at -18 ºC. A stainless-steel mesh was used as the counter electrode.

Laser ablation: An adaptive laser ablation procedure was implemented, which was able to accommodate the various device layouts. The ablation platform used in this work was a HL40-5g, from Full Spectrum Laser LLC, which includes a 40 W CO 2 laser operated in raster mode at 500 ppi and 100 % power. In these conditions, the devices were properly trimmed without producing damage by overheating the materials, since in the raster mode the laser only fires for few ms in each location.

The cutting path was adapted to each particular device by first scanning (Konica Minolta C284 PS flatbed scanner) each VPP PEDOT pattern , and then directly drawing the desired laser path on the digitalized image of each geometry using Photoshop. The result of the drawing procedure was then transformed in a bitmap file (.bmp), which can be interpreted by the printer to command the laser path.

Final adjustments, such as printing direction, scaling and thresholding (grey level selected for black & white transition) were directly performed in the laser control software (Full Spectrum RetinaEngrave3D). µCP: The PDMS precursors (after mixing and de-gazing) were polymerized on top of the template (silicon wafer with SU-8 pattern). The PDMS stamp was removed from the template and then treated for 30 s in an O 2 plasma.

The different oxidant solutions were spin coated at 2000 rpm/500 rpms -1 for 30 s on glass slides. By stamping the PDMS stamp on the iron tosylate spincoated glass slide, the PDMS stamp got inked. The oxidant inked stamp can then pattern the PVDF and PVA substrates using μCP. The oxidant pattern underwent VPP in the following step.

Syringe-based printing: Oxidant solutions were deposited using a syringe-based printer (Seraph Robotics Inc. Fab@Home Model 3), using a 200 µm-diameter nozzle (straight cannula, blunt end tip, Fisnar Inc.). The different structures were printed using a deposition rate of 3.9310 -5 m 3 s -1 and translation speed of 3 mms -1 at 21 °C. The printed oxidant pattern underwent VPP in the following step.

Contact angle (θ) measurements were taken with a camera just after the deposition of the droplet on the substrate. Measurements were made on three samples.

Actuation test: Ivium compactstat or IviumStat (Ivium technologies) was used to electrically address the actuators. Gold coated kelvin clips were used to directly contact the actuators. The displacement was recorded with a mobile phone camera.

Strain ε: The strain is calculated from the following expression for the strain at any location along the actuator length L [START_REF] Alici | An Effective Modelling Approach to Estimate Nonlinear Bending Behaviour of Cantilever Type Conducting Polymer Actuators[END_REF][START_REF] Gaihre | Synthesis and Performance Evaluation of Thin Film PPy-PVDF Multilayer Electroactive Polymer Actuators[END_REF] ; ε (x) = 2 ℎ (𝐿-𝑥) 2 𝐿 4 𝑦𝑡𝑖𝑝; x=0; where the variable x starts from the fixed end of the actuator, h and ytip are the total thickness of the actuator and tip displacement of the actuator in the transverse direction, respectively.

ASSOCIATED CONTENT

Supporting information

Non-essential figures and descriptions that complement the findings and discussion of the manuscript: PEDOT micropatterned surfaces by micro-contact printing on different substrates (PDF); Video of a laser cut actuating hand.

Figure 1 .

 1 Figure 1. Schematic showing CP trilayer actuation. The dark layers represent the CP layers, which sandwich an ionic conductive polymer electrolyte membrane, shown in white. Three different actuation states are presented: the unbiased state (center), and the positively and negatively biased states (left and right, respectively). Note that the device is symmetric and that the applied potential defines the direction.

Figure 2 .

 2 Figure 2. Vapor phase polymerization a) Schematic of the process, b) Thickness, and c) conductivity of the resulting PEDOT layer as a function of PEGM-PEGDM content used in the oxidant solution.

Figure 3 .

 3 Figure 3. Contact angles of the oxidant solution on several substrates, as a function of the PEGM-PEGDM content in the solution.

Figure 4 .

 4 Figure 4. Process flow of the fabrication of conjugated polymer actuators: µ-contact printing and syringe-based printing techniques for use with vapor phase polymerization. 1) The PDMS stamp is pressed onto the oxidant solution deposited by spin-coating on a glass slide. 2) The PDMS stamp is removed from the substrate and the

Figure 5 .

 5 Figure 5. µCP PEDOT patterns. a) Pattern on PVA obtained after 30 min of VPP onto a µCP-patterned oxidant layer with 10 % PEGM-PEGDM, b) The µCP pattern obtained with the same process but with 30 % PEGM-

Figure 6 .

 6 Figure 6. a) A schematic of the patterning process for the fabrication of a CP trilayer actuator device. The bottom electrodes were deposited by syringe-based printing of the oxidant solution on glass, followed by VPP of PEDOT.

Figure 7 .

 7 Figure 7. a) Scanning electron microscopy image of the trilayer structure cross-section. b) A 500 x 2000 µm 2 trilayer bending beam CP actuator of with its contact pad patterned using laser ablation. c) Five-millimeter-wide patterned hands. d) Current signal from the actuation of the hand at a frequency of 1 Hz and potential amplitude of ± 2 V. Video S-1 shows the actuation of one hand.

Figure 8 .

 8 Figure 8. a) An array of syringe printed, individually controllable, flexible actuators. b) Juxtaposition of two photographs showing the extreme states of the actuated trilayer device with an applied potential ±1 V at 0.025 Hz (presence of PPy layer). c) Top view of a device with two individually controllable digits. The device consists of two juxtaposed trilayer actuators, connected at the bottom by a ~9 mm long segment of the polymer electrolyte layer, cut from the array of actuators shown in (a) using laser ablation.

).[START_REF] Ugur | Low-Dimensional Conduction Mechanisms in Highly Conductive and Transparent Conjugated Polymers[END_REF][START_REF] Lee | Heavily Doped poly(3,4-Ethylenedioxythiophene) Thin Films with High Carrier Mobility Deposited Using Oxidative CVD: Conductivity Stability and Carrier Transport[END_REF] The acrylate oligomers in our oxidant solution promote adhesion of the CP layer to acrylic

Funding Sources

This study was financially supported by Linköping University, COST Action MP1003 ESNAM (European Scientific Network for Artificial Muscles), the Swedish Research Council (VR -2010-6672, 2014-3079, 2015-03298), the Knut & Alice Wallenberg Stiftelse (LiU-2010-00318 & LiU-2012-01361), and the EU FP7 Marie Curie action IEF (625923 POLYACT).

ACKNOWLEDGMENT

The authors acknowledge Anthony Turner for his support.

individually fabricated electrochemical devices such as soft actuators, flexible batteries, and stretchable components for bioelectronics and soft robotics.

Experimental

Materials: The oxidant solution, 40wt% Fe(Tos) 3 solution in ethanol/Butanol mixture was obtained from Heraeus corporation. Pyrrole was obtained from Sigma-Aldrich, distilled and stored at -18 °C prior to use. Azobisisobutyronitrile (AIBN, Sigma-Aldrich) was recrystallized in methanol prior to use. Cyclohexanone (Acros, 99.8%), poly(ethylene glycol)dimethacrylate.

(PEGDM, Mn = 750 g mol -1 , Aldrich), poly(ethylene glycol) methyl ethermethacrylate (PEGM, Mn = 475 g mol -1 ), polyvinylalcohol (PVA, Mowiol 4-88, Mw≈31000 g.mol -1 ) were purchased from Sigma-Aldrich. Nitrile butadiene rubber (NBR) with 44 wt% acrylonitrile content was obtained from Lanxess.

3,4-azobisisobutyronitrile (AIBN, Sigma-Aldrich) was recrystallized in methanol prior to use. PVA deposition: A 10 wt.% PVA solution was prepared by dissolving PVA in water while stirring for 24 h at 95 ºC. The PVA layer was deposited on a glass substrate from the PVA solution using a spin coater (Ramp 500 rpms -1 , 500 rpm for 30 s). A curing step of 5 minutes at 50 ºC was applied to dry the film. PEO-NBR semi-IPN synthesis [START_REF] Festin | Robust Solid Polymer Electrolyte for Conducting IPN Actuators[END_REF] PEO/NBR solid polymer electrolyte composition: PEO-NBR 50/50 semi-IPN film was obtained by an in-situ polymerization. First the linear NBR solution was prepared by dissolving NBR in cyclohexanone (1:5 weight ratio). After complete dissolution, PEO network precursors (50 wt.% vs NBR) were introduced by adding PEGM monomer (75 wt.% of the PEO network), PEGDM crosslinker (25 wt.% of the PEO network)

AUTHOR INFORMATION

Corresponding Author

Author Contributions

The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript.


Philippe Joos

Jonathan Porée

Hervé Liebgott

Didier Vray

Mathilde Baudet

Julia Faurie

François Tournoux

Guy Cloutier

Barbara Nicolas

Damien Garcia
email: garcia.damien@gmail.com

High-Frame-Rate Speckle-Tracking Echocardiography

Keywords: Cardiac imaging, diverging waves, high-framerate echocardiography, motion compensation (MoCo), speckletracking echocardiography (STE), ultrafast ultrasound

à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

I. INTRODUCTION

E CHOCARDIOGRAPHY is one of the most widespread modalities for cardiovascular imaging due to its high temporal resolution and low cost, because it is a safe real-time diagnostic imaging modality [START_REF] Vray | Ultrasound medical imaging[END_REF]. Speckle-tracking echocardiography (STE) is a quantitative method for assessing the dynamics of cardiac motion. It allows the measurement of myocardial velocities and deformations in the short-axis and apical views and provides valuable information on cardiac synchrony and function [START_REF] Geyer | Assessment of myocardial mechanics using speckle tracking echocardiography: Fundamentals and clinical applications[END_REF]. When STE is used to derive strains and strain rates, this technique is also referred to as "strain imaging" [START_REF] Gorcsan | Echocardiographic assessment of myocardial strain[END_REF]. STE is now recognized as a quantitative key tool in clinical cardiac research. STE algorithms generally use a block-matching approach to track the speckles in a sequence of 2-D B-mode (grayscale) images [START_REF]Principles and different techniques for speckle tracking[END_REF]. In the current clinical practice, it is admitted that a frame rate of 50-80 frames/s returns optimal conditions for speckle tracking in the resting heart beating at ∼70 bpm [START_REF] Voigt | Definitions for a common standard for 2D speckle tracking echocardiography: Consensus document of the EACVI/ASE/Industry Task Force to standardize deformation imaging[END_REF]. In some situations, such as for the evaluation and management of coronary artery disease, it can be recommended to increase the heart rate during an echocardiographic examination, i.e., perform a stress echocardiography [START_REF] Marwick | Stress echocardiography[END_REF]. In a stressed myocardium, the mechanical events become shorter; the acquisition frame rate should thus be increased (probably proportionally) with the heart rate (up to 120-140 bpm) [START_REF] Voigt | Definitions for a common standard for 2D speckle tracking echocardiography: Consensus document of the EACVI/ASE/Industry Task Force to standardize deformation imaging[END_REF]. Acquiring the whole left ventricular myocardium at such high frame rates is challenging with the conventional imaging systems. For this reason, no consensus has been reached about the incorporation of STE in routine stress echocardiography. Stress echocardiography could thus benefit from high-frame-rate ultrasound imaging (100-500 frames/s) [START_REF] Cikes | Ultrafast cardiac ultrasound imaging: Technical principles, applications, and clinical benefits[END_REF], [START_REF] Andersen | High-frame-rate deformation imaging in two dimensions using continuous speckle-feature tracking[END_REF].

Several techniques have been proposed in the past few years to increase frame rate in transthoracic cardiac ultrasound imaging while keeping high-quality images. Transmit schemes based on focused or unfocused beams have been introduced. In the multiline transmit (MLT) technique, several focused beams are transmitted into different directions [START_REF] Mallart | Improved imaging rate through simultaneous transmission of several ultrasound beams[END_REF]. Combined with multiline acquisition, MLT frame rate can be further increased [START_REF] Tong | Multitransmit beam forming for fast cardiac imaging-Experimental validation and in vivo application[END_REF]. MLT-based tissue Doppler imaging at >200 frames/s was recently reported [START_REF] Tong | Wide-angle tissue Doppler imaging at high frame rate using multi-line transmit beamforming: An experimental validation in vivo[END_REF]. Ultrafast cardiac ultrasound imaging is also possible with diverging waves [START_REF] Hasegawa | High-frame-rate echocardiography using diverging transmit beams and parallel receive beamforming[END_REF]- [START_REF] Porée | High-frame-rate echocardiography using coherent compounding with Doppler-based motion-compensation[END_REF]. To preserve contrast and spatial resolution in unfocused wave imaging, coherent compounding of images derived from different steering-angle transmits is essential since the individual images are of poor quality. Coherent compounding corrects the phase delays related to the transmit and receive travel times. It improves the image quality of motionless or slow-moving tissues. However, if the motion of fast-moving scatterers is neglected, it may cause destructive interferences and, in turn, degrade contrast and resolution [START_REF] Wang | Motion artifacts of extended high frame rate imaging[END_REF], [START_REF] Denarie | Coherent plane wave compounding for very high frame rate ultrasonography of rapidly moving targets[END_REF]. Indeed, large motions can generate substantial phase delays, thus producing noncoherent summation. Consequently, a traditional compounding approach is not adapted to cardiac imaging, especially under pharmacological stress or physical exertion. The integration of motion compensation (MoCo) in the compounding process has been shown to ensure coherent summation and thus improve the image quality significantly. MoCo is based on the estimation of axial [START_REF] Denarie | Coherent plane wave compounding for very high frame rate ultrasonography of rapidly moving targets[END_REF] or 2-D [START_REF] Gammelmark | 2-D tissue motion compensation of synthetic transmit aperture images[END_REF] motion between the successive tilted images. The signals are then rephased before being summed coherently. It has been shown in [START_REF] Porée | High-frame-rate echocardiography using coherent compounding with Doppler-based motion-compensation[END_REF] that radial (axial) motion only needs to be compensated in phased-array cardiac imaging since the cross-range resolution is low compared with that of the radial resolution. It has also been demonstrated in the same study that a triangle transmit sequence (i.e., steering angles increasing then decreasing linearly) is more efficient than linear or alternate transmission strategies for MoCo based on Doppler estimation. The triangle sequence has the advantage to sum the main lobes coherently and the side lobes incoherently. To apply MoCo, the motion is assumed constant during N successive transmits, providing one tissue Doppler image: one high-quality compound image is then obtained by summing the N motion-compensated complex envelopes.

Speckle tracking is a suitable method for myocardium displacement measurement [START_REF]Principles and different techniques for speckle tracking[END_REF]. This technique, based on the local conservation of the speckle patterns, cannot be applied if artifacts or poor image quality compromise speckle recognition from one frame to the next one. As illustrated in the human left ventricle [START_REF] Porée | High-frame-rate echocardiography using coherent compounding with Doppler-based motion-compensation[END_REF], coherent compounding can produce major signal losses if MoCo is not integrated. This is particularly true when the axial myocardial displacements are large, mostly during peak diastole (e ), and peak atrial (a ) or ventricular (s ) systoles. On the other hand, the speckle patterns are well preserved when MoCo is taken into consideration, thus enabling motion estimation based on the tracking of these patterns. We here hypothesized that high-frame-rate high-quality echocardiography based on MoCo allows quantification of fast cardiac motions. Speckle-tracking methods are most commonly based on block matching with normalized cross correlation [START_REF]Principles and different techniques for speckle tracking[END_REF], followed by a subpixel refinement. Subpixel fine tuning is a key step when the frame-to-frame displacements are less than that of the pixel size. Three standard methods were tested in this paper. The objective of this paper was twofold: 1) show that accurate STE can be obtained from standard block-matching with high-frame-rate echocardiography based on diverging wave imaging and 2) determine which subpixel refinement is most appropriate. STE was first validated in vitro in a rotating disk with normal to subnormal myocardial speeds. High-frame-rate STE of the left ventricular myocardium was then produced in vivo in 10 healthy volunteers. The in vivo data were acquired with a Verasonics research scanner and the local myocardial displacements were determined using speckle tracking with three different algorithms for subpixel refinement. The global longitudinal strain (GLS) was also determined as it is a robust marker of left ventricular systolic function [START_REF] Reisner | Global longitudinal strain: A novel index of left ventricular systolic function[END_REF]. The GLS waveforms measured by highframe-rate echocardiography (500 frames/s) were compared against those obtained with a state-of-the-art clinical General Electric GE scanner (80 frames/s) and commercial workstation (EchoPAC, GE).

II. METHOD

A. Motion-Compensated High-Frame-Rate Ultrasound

The ultrasound in-phase and quadrature components (I/Q) signals were acquired with a Verasonics research scanner (V-1-128, Verasonics Inc., Redmond, WA, USA) and a 2.5-MHz cardiac phased-array transducer (ATL P4-2, 64 elements). The acquisition parameters are reported in Table I. To be consistent with [START_REF] Porée | High-frame-rate echocardiography using coherent compounding with Doppler-based motion-compensation[END_REF], we applied series of 36 tilted 90°-wide diverging waves, with virtual sources located behind the probe (maximal distance = 1.1 cm), to generate high-contrast high-resolution images with the MoCo approach during the compounding process. The successive transmit tilt angles (ranged between -16°and 16°) were arranged triangularly (i.e., increasing then decreasing linearly) to sum the main lobes coherently and the side lobes incoherently, as in [START_REF] Porée | High-frame-rate echocardiography using coherent compounding with Doppler-based motion-compensation[END_REF]. An image rate of 500 images/s was reached using a 36-sample sliding window with 75% overlap (PRF/36×4 = 4500/36×4 = 500 FPS). The MoCo beamforming protocol was similar to that described in [START_REF] Porée | High-frame-rate echocardiography using coherent compounding with Doppler-based motion-compensation[END_REF]: to generate one compound image, the I/Q signals were summed coherently after delay-and-sum and Dopplerbased MoCo. Speckles were tracked on the log-compressed real envelopes (containing 256 radial scanlines, with 500 axial samples each). A clinical GE Vivid q scanner was also used with another 2.5-MHz phased-array transducer (M4S-RS) for the in vivo validation (see Section II-D, In Vivo Experiments).

B. Speckle-Tracking Echocardiography

STE was achieved by tracking the speckle patterns using block matching with subsequent subpixel refinement. Since the displacement fields were expected to be smooth, with relatively low-spatial step gradients, we tracked the speckles on lowfrequency real envelopes (i.e., amplitude of I/Q signals). This I/Q-based approach also had the methodological advantage to be potentially less sensitive to clutter and to halve data amount (by using quadrature sampling) when compared to RF-based tracking. To determine the motion with a one-pixel precision (whose size was half-wavelength × 0.35°), we first used the standard method based on the normalized cross correlation calculated in the Fourier domain. Postprocessing parameters are reported in Table I. The real-envelope images (before scan conversion) were divided into small regions of interest whose dimension (32 × 32) corresponded to 1 cm in the radial direction, and 11°in the cross-range direction. We worked with ensembles of 20 consecutive images, under the assumption that the motion remained unchanged during the time of the successive acquisitions (4.4 ms), to calculate the average of 19 cross correlation matrices (ensemble correlation, see [START_REF] Raffel | Image evaluation methods for PIV[END_REF]). Peak detection of the averaged normalized cross correlation provided the displacements with a pixel precision. Subpixel precision of the displacement estimates was then obtained through three different methods as follows.

1) Parabolic Peak Fit of the Cross Correlation: The correlation was assumed to follow a paraboloid about the peak. Three-point stencils were used in the two directions to locate the correlation peak at a subpixel level [19, p. 160] 2) Phase Correlation Method: The phase angles of the normalized cross-power spectrum were fit to a plane using a robust linear regression [20, eq. ( 3)]. The two slopes of the plane provided the two components of the displacement vectors. 3) Differential Optical Flow: The Lucas-Kanade method was used to solve the optical flow equations on the real envelopes. The ensemble included in the overdetermined linear system contained 19 image windows. Hanning weights were assigned in the resulting weighted leastsquares problem [21, eq. ( 8)]. The velocity vectors derived from STE were postprocessed with a robust and unsupervised regularizer based on discrete cosine transforms [START_REF] Garcia | Robust smoothing of gridded data in one and higher dimensions with missing values[END_REF]. This smoother was validated in 2-D and 3-D velocity vector fields containing noisy and spurious values [START_REF] Garcia | A fast all-in-one method for automated post-processing of PIV data[END_REF]. To avoid subjective smoothing, the regularizing parameter was selected automatically by minimizing the generalized cross-validation GCV score [START_REF] Garcia | Robust smoothing of gridded data in one and higher dimensions with missing values[END_REF].

C. In Vitro Experiments

In vitro experiments were carried out in a 10-cm-diameter rotating disk connected to a step motor. The scales of the experiment matched those observed in the echocardiographic apical view: the upper edge was 2 cm far from the probe, and the scan-depth was 15 cm. The acoustic properties of the disk were tissue like, with the following composition: agar 3%, Sigmacell cellulose powder 3%, glycerol 8%, and water. The outer speeds (velocity amplitudes at the circumference) ranged from 1 to 35 cm/s with a 1.1-cm/s step (maximum angular velocity of 7 rad/s with a 0.2 rad/s step). The angular velocities were chosen to replicate tissue speeds of the left myocardium which can reach values up to 30 cm/s in athletes [START_REF] Caso | Pulsed Doppler tissue imaging in endurance athletes: Relation between left ventricular preload and myocardial regional diastolic function[END_REF]. The STE-derived velocity vectors were compared with the groundtruth velocity vector field given by the radius and the rotational speed of the disk. Absolute velocity errors were calculated both in radial and cross-range directions and normalized by the outer speeds of the disk. The medians of the normalized errors were reported for a given angular velocity.

D. In Vivo Experiments

The aim of this in vivo study was to investigate how high-frame-rate STE compared with state-of-the-art clinical echocardiography for assessing global myocardial deformation. An experienced physician acquired apical four-chamber views of the left ventricle with a GE Vivid q scanner (GE Healthcare) and the Verasonics scanner, successively. The 10 healthy volunteers aged 20-40 years were enrolled in the in vivo protocol approved by the human ethical review committee of the CRCHUM (Research Center of the University of Montreal Hospital, Montreal, QC, Canada). The GE imaging sequence was a conventional sequential focusing approach at 60-80 frames/s. The Verasonics scanning sequence and the MoCo beamforming approach are described in Section II-A. Since the I/Q signals issued from the diverging waves were beamformed offline, the on-screen display with the Verasonics was of poor quality (no MoCo, low refresh rate). With the Verasonics scanner, the location and orientation of the probe were thus adjusted using a prior sequential focused sequence before switching to the high-frame-rate acquisition. From these 10 pairs (GE + Verasonics) of acquisitions, we computed the GE-and Verasonics-derived GLS. The GLS is a prognostic marker of the global left ventricular systolic function measured clinically by STE from a long-axis apical view [START_REF] Kalam | Prognostic implications of global LV dysfunction: A systematic review and meta-analysis of global longitudinal strain and ejection fraction[END_REF]. It reflects the relative longitudinal contraction (in percent) of the myocardium. The GLS peak is known to be around -20% in normal subjects [START_REF] Yingchoncharoen | Normal ranges of left ventricular strain: A meta-analysis[END_REF]. The GE-derived GLS were determined by a physician using an EchoPAC workstation (GE Healthcare) with the proprietary speckle-tracking technique. The Verasonics-derived GLS was measured after STE with the three abovementioned subpixel methods as follows. For a given subject, the endocardium was delineated manually in the first B-mode image of the high-frame-rate series under the supervision of a physician. This sampled contour was tracked automatically, from frame-to-frame, using the three abovementioned subpixel techniques. The instantaneous GLS (in percentage) was estimated as GLS(t) = 100[L(t)max(L)]/ max(L) where L(t) is the longitudinal endocardial length at time t. Note that we used the maximum length of the endocardium instead of its length at end-diastole since we did not acquire the electrocardiogram signal when scanning with the Verasonics. This approximation does not affect GLS significantly in normal patients. The drifts of the strain curves were corrected assuming that the length of the left ventricle should return to its original length after a complete heart cycle. In clinical STE, drift correction is recommended to remove cumulative errors. Drift was corrected by affine regression. The GLS peaks determined by high-frame-rate echocardiography (Verasonics) were compared with those returned by the EchoPAC workstation. The four groups (one GE-derived + three Verasonics-derived) were compared using a multiple pairwise comparison test with the Bonferroni correction (MAT-LAB, Statistics Toolbox, Mathworks Inc.).

III. RESULTS

A. In Vitro

The velocity vector images of the disk (see Fig. 1) show that STE was able to uncover the rigid rotation, with the three subpixel methods, when MoCo was involved. As a comparison, Fig. 2 illustrates that MoCo was essential for speckle tracking, especially in the presence of large motions.

The estimated velocity fields with MoCo were consistent with the ground-truth fields, but a bias was observed (the center was upward) likely due to the degradation of the crossrange resolution with depth. As shown in Fig. 3, the normalized cross-range errors were higher than the normalized radial errors (lower bound = 10% versus 4%, Fig. 3). The phase correlation performed better than did the other methods in vitro (Fig. 3; radial: 4.9% ± 0.7%; and cross-range 12.4% ± 0.7%). When MoCo was not integrated in the compounding process, errors on the velocity estimations reached 45% in the radial direction when the rotation speed was high, which confirms that MoCo is needed to get high-frame-rate STE. Without MoCo, the speckle patterns were indeed not preserved due to the presence of destructive interferences (see Fig. 2). The optical flow method also returned small errors in the radial direction (5.7% ± 0.8%) but produced the greatest errors in the cross-range direction. The peak fitting approach returned the largest errors in the radial direction, especially when the velocities were small. Peak fitting by interpolation of the cross correlation peak is indeed subject to significant inaccuracy, especially when the frame-to-frame displacements are less than one pixel in magnitude [START_REF] Christensen | The influence of peak-locking errors on turbulence statistics computed from PIV ensembles[END_REF]. As a side note, speckle tracking with GE-derived B-mode images, obtained at maximum frame rate (70 frames/s), failed with the three methods for large motions. The normalized errors in the crossrange direction were between 20% and 50% beyond a rotation speed of 5 rad/s.

B. In Vivo

Fig. 4 shows left ventricular velocity vector images of one volunteer during systole (left column) and diastole (right column). Velocity vectors from peak fitting and optical flow have similar directions, but smaller amplitudes were observed with peak fitting. The phase-based approach failed to detect cardiac motion. These observations were repeated throughout the whole cardiac cycle, as can be seen in the movies of the supplementary content.

The GLS waveforms of the same subject are depicted in Fig. 5 and compared with that obtained from the GE scanner and workstation. Consistently, with the velocity vector images of Fig. 4, subpixel refinement with optical flow returned the best match. Peak fitting and phase correlation led to substantial underestimations. This was observed in 10 volunteers, as revealed by the GLS systolic peaks (see Fig. 6). The optical flow and GE methods returned values in the normal range (around -20%), whereas the GLS peaks obtained with peak fitting and phase correlation were in the subnormal range. Although a larger variance was observed in the optical flow method in comparison with GE, their means were not significantly different ( p value = 0.33). The other pairwise comparisons indicated significant differences of the means ( p value < 10 -5 ). The Bland-Altman statistics (Verasonics GLS -GE GLS, mean ±2 std) were: GE versus 1) peak fitting: 7.2% ± 3.6%; 2) phase correlation: 14.7% ± 4.3%; and 3) optical flow: 2.2% ± 4.7%. These results denote that phase correlation and peak fitting are likely not adapted for in vivo high-frame-rate STE.

IV. DISCUSSION

We introduced high-frame-rate STE based on steered diverging waves, MoCo, and speckle tracking by block matching. We obtained STE at 500 frames/s, which is around 6 times higher than in conventional clinical scanners. The in vitro study showed that high-tissue velocity amplitudes (up to 30 cm/s) can be measured with our method. The pilot in vivo study illustrated that the GLSs determined by high-frame-rate STE in 10 subjects were consistent with those measured with a commercial workstation. At very high frame rates, local frame-to-frame displacements can be very small. Subpixel motion estimation is thus a critical aspect. Three basic different schemes for subpixel refinement in speckle tracking were tested. In our in vivo study, a subpixel motion estimator through an optical flow method returned the best outputs. To sum up, this paper demonstrates that:

1) MoCo is a necessary condition for myocardial speckle tracking when coherent compounding is involved. 2) STE of the myocardium is feasible at very high frame rates. 3) Robust algorithms for subpixel motion estimation are of key importance when dealing with in vivo data. These aspects are discussed in the following.

A. Significance of Motion Compensation for Speckle Tracking in High-Frame-Rate Echocardiography

Assessment of the myocardial dynamics by speckle tracking is possible only if the speckle patterns are well preserved, which can be challenging at very high frame rates. Andersen et al. [START_REF] Andersen | High-frame-rate deformation imaging in two dimensions using continuous speckle-feature tracking[END_REF] have tackled this problem by introducing a multistep tracking method, including spatial and temporal filtering, detection of strong-intensity speckles, constrained feature tracking, and final smoothing. They used a 16:1 parallel receive ("explososcanning," [START_REF] Shattuck | Explososcan: A parallel processing technique for high speed ultrasound imaging with linear phased arrays[END_REF]) to obtain long-axis views of the left ventricle at 500 frames/s. This composite-tracking process might have been necessary to deal with the relatively low contrast of their images (no coherent compounding was used). In this original feasibility study, they tested their approach on 10 subjects and confirmed that speckle tracking is possible in high-frame-rate echocardiography. In our study, we obtained wide-sector scans of high-quality images of the four cardiac chambers at 500 frames/s. As explained earlier, axial motion was compensated during coherent compounding Fig. 4. Systolic (left column) and diastolic (right column) motion fields of the left myocardium in one healthy volunteer. From top to bottom: velocity vector images derived from three different subpixel techniques based on: 1) peak fitting; 2) phase correlation; and 3) optical flow. Fig. 5. GLS waveform in one healthy volunteer (same as in Fig. 4). GLS was obtained from motion estimation measured by peak fitting, phase correlation, and optical flow. The GLS provided by the GE clinical scanner is represented as a reference.

to discard the destructive interferences that can be generated by the large movements of the myocardium. To this end, MoCo was carried out using an original method developed by Porée et al. [START_REF] Porée | High-frame-rate echocardiography using coherent compounding with Doppler-based motion-compensation[END_REF] and based on tissue Doppler. In the absence of MoCo, significant signal losses were discernible in the B-mode images see [START_REF] Porée | High-frame-rate echocardiography using coherent compounding with Doppler-based motion-compensation[END_REF]Fig. 12], especially during peak diastole and systole, thus making myocardial speckle tracking not viable. In this paper, we demonstrated both in vitro and in vivo that preservation of the speckle patterns through MoCo allowed accurate cardiac motion estimation at high frame rates with a standard subpixel block-matching algorithm. This was confirmed by the consistent GLS waveforms obtained with the optical-flow motion estimator in 10 volunteers using a Verasonics research scanner. Hence, MoCo is a necessary condition for myocardial speckle tracking when coherent compounding is required. When less (or no) compounding is done, the adverse effect of motion might be less detrimental and might, therefore, have less impact on speckle tracking. It is expected that a compromise must be made in terms of image and tracking quality. With regard to the MLT approach, since it is compounding free [START_REF] Mallart | Improved imaging rate through simultaneous transmission of several ultrasound beams[END_REF], [START_REF] Tong | Multitransmit beam forming for fast cardiac imaging-Experimental validation and in vivo application[END_REF], MoCo is not needed. It would be of interest to investigate to which extent the receipt crosstalks inherent in MLT may affect speckle tracking.

B. Comparison of the Speckle-Tracking Algorithms

Three basic subpixel block-matching methods were tested, with similar input parameters, both in vitro and in vivo, i.e., similar subwindow size, ensemble length, and validation. Significant differences were observed between the in vitro and in vivo findings, leading to contradictory conclusions. The phasebased approach was the most accurate in vitro, regardless of the rotation speed. However, it was ineffective in vivo. The rotating disk setup provided high-contrast high-SNR B-mode images, was free of out-of-plane motions, and the movements were rigid (no deformation) and steady (no acceleration). These ideal conditions were obviously not met in vivo. Furthermore, the in vivo images were marred by artifacts classically encountered in medical ultrasound imaging, and mostly related to propagation path and attenuation. These disparities likely explain, in large part, the conflicting results observed in our experiments, especially regarding the phase correlation. Phasebased algorithms are indeed known to be very sensitive to noise [START_REF] Foroosh | Extension of phase correlation to subpixel registration[END_REF]. According to our results, it is thus likely that phase-based approaches are poorly adapted for high-framerate echocardiography. A number of numerical techniques have been introduced to mitigate the adverse effects of noise for phase correlation with least-squares fitting (see [START_REF] Hoge | A subspace identification extension to the phase correlation method[END_REF], [START_REF] Ren | High-accuracy sub-pixel motion estimation from noisy images in Fourier domain[END_REF]). In our study, however, we only focused on the comparison of three different motion trackers in their most basic form. Note that the transverse oscillation approach, also a phasebased technique, has been successfully validated in vivo with large-aperture (linear) arrays [START_REF] Salles | 2-D arterial wall motion imaging using ultrafast ultrasound and transverse oscillations[END_REF], [START_REF] Hansen | Analysis of systolic backflow and secondary helical blood flow in the ascending aorta using vector flow imaging[END_REF]. In the same direction, a recent theoretical study described the optimal conditions to estimate the cross-range motion using transverse oscillations in cardiac phased-array imaging [START_REF] Heyde | Evaluation of the transverse oscillation technique for cardiac phased array imaging: A theoretical study[END_REF]. Since their findings were not supported by in vivo data, no explicit conclusions can be pronounced regarding the clinical utility. Of note, one in vivo case for cardiac motion in echocardiography with transverse oscillations was analyzed by Alessandrini et al. [START_REF] Alessandrini | A new technique for the estimation of cardiac motion in echocardiography based on transverse oscillations: A preliminary evaluation in silico and a feasibility demonstration in vivo[END_REF]. The reported global strain values, however, were much below the normal ranges. Further in vivo studies are thus required to check the clinical reliability of this approach. The method based on peak fitting constantly underestimated the displacements both in vitro and in vivo. This can be explained by the well-documented "peak-locking effect," the tendency of peak fitting to bias toward integral pixel values [START_REF] Cholemari | Modeling and correction of peak-locking in digital PIV[END_REF]. In our study, pointwise frame-to-frame displacements were all less than 0.5 pixels in the cross-range direction; the peak-locking effect thus induced a consistent bias toward zero. Although other fitting models exist (paraboloid, Gaussian, centroid, etc.,), they are also all sensitive to the peak-locking effect. In our study, the differential optical flow approach returned the most accurate GLS waveforms in 10 subjects (see Figs. 4 and5), and they were very consistent with those obtained by the clinical GE scanner and the EchoPAC workstation, although some underestimation and larger variance were noticed. This further confirms that the myocardial speckles were well preserved during diverging wave imaging with MoCo beamforming.

C. Possible Improvements of the Optical Flow Method

The use of high-frame-rate echocardiography allowed us to implement algorithms based on large slow-time ensembles (of length 20): 1) ensemble correlation before peak fitting; 2) ensemble phase correlation; or 3) overdetermined linear systems in the optical flow approach. As already discussed, a differential optical flow method provided the best results. In this paper, we used a local Lucas-Kanade approach in its simplest form (locally rigid translations). Parametric models could be integrated to potentially improve motion estimation, such as those assuming locally affine motions [START_REF] Ju | Skin and bones: Multilayer, locally affine, optical flow and regularization with transparency[END_REF]. In particular, this approach has long been used in vascular elastography [START_REF] Maurice | On the potential of the Lagrangian estimator for endovascular ultrasound elastography: In vivo human coronary artery study[END_REF], [START_REF] Naim | Characterisation of carotid plaques with ultrasound elastography: Feasibility and correlation with high-resolution magnetic resonance imaging[END_REF]. Since the myocardial velocity field is smooth, a global regularized method [START_REF] Tarnec | A proof of convergence of the Horn-Schunck optical flow algorithm in arbitrary dimension[END_REF] could also increase the robustness of the estimation. In addition, because tissue Doppler is given by the MoCo process, it could be combined with the optical flow measurement in a least-squares regularized problem as in [START_REF] Tavakoli | Tissue Doppler imaging optical flow (TDIOF): A combined B-mode and tissue Doppler approach for cardiac motion estimation in echocardiographic images[END_REF]. A supplementary constraint based on tissue Doppler can very likely reduce biases. Finally, the addition of physiological constraints or deformation models [START_REF] Queirós | Fast left ventricle tracking using localized anatomical affine optical flow[END_REF] could further reduce errors due to out-of-plane motions, for example.

D. Global Longitudinal Strain (GLS)

The GLS waveforms and peaks were concordant with those returned by a clinical ultrasound scanner (Figs. 5 and6). We choose GLS as clinical index to validate our in vivo results since it has become widely accepted in strain imaging for assessing the systolic left ventricular function. Due to significant intervendor variability in strain images, it has been recently reported that GLS is the only myocardial strain parameter which may be safely used in routine clinical practice [START_REF] Farsalinos | Head-to-head comparison of global longitudinal strain measurements among nine different vendors: The EACVI/ASE intervendor comparison study[END_REF]. It would be of interest to test our approach for determining longitudinal strains locally to offer high-framerate strain imaging. This would be particularly relevant for simultaneous strain imaging in all four cardiac chambers, where wide deep sectors are required at the expense of frame rate [START_REF] Addetia | Simultaneous longitudinal strain in all 4 cardiac chambers: A novel method for comprehensive functional assessment of the heart[END_REF]. High-frame-rate strain imaging can also be of importance in stress echocardiography where heart beats are around 120 bpm. We plan to address these topics in a future study with an MRI-ultrasound-compatible in vitro phantom [START_REF] Saloux | Heart phantom assembly[END_REF] which reproduces myocardial shortening, torsion, and contraction (lengthening, untwisting, and dilation) during systole (diastole).

E. Volumetric Three-Component STE and Volumetric GLS

To fully characterize cardiac motion and deformation, volumetric three-component velocity vector imaging (STE) will be the logical continuation of this 2-D study. Since its temporal resolution is limited, 3-D echocardiography is currently restricted by the need of stacking several small scan volumes acquired during consecutive heart beats. This limitation makes 3-D STE not clinically compliant since it requires time-demanding acquisitions and supervised postprocessing. High-frame-rate 3-D cardiac imaging will be needed to attain sufficient temporal and spatial resolutions in a single heartbeat. Different potential strategies are worth mentioning: 1) multiplane transmits [START_REF] Chen | Feasibility of multiplane-transmit beamforming for real-time volumetric cardiac imaging: A simulation study[END_REF], a generalization of the MLT method; 2) row-column-addressed arrays [START_REF] Christiansen | 3-D imaging using row-columnaddressed arrays with integrated apodization-Part II: Transducer fabrication and experimental results[END_REF]; 3) 2-D sparse arrays [START_REF] Roux | Wideband 2-D array design optimization with fabrication constraints for 3-D US imaging[END_REF]; 4) synthetic aperture imaging with a 1024-element 2-D transducer array [START_REF] Rasmussen | Comparison of 3-D synthetic aperture phased-array ultrasound imaging and parallel beamforming[END_REF], [START_REF] Provost | 3D ultrafast ultrasound imaging in vivo[END_REF]. Whether these different procedures adequately preserve the speckle patterns for effective speckle tracking has not been investigated. To obtain highframe-rate high-quality volumetric echocardiography, we will update our MoCo strategy for 3-D using steered spherical diverging waves.

V. SUMMARY AND CONCLUSION

In this paper, we presented an innovative method based on the combination of MoCo and speckle tracking to perform high-frame-rate STE of the left ventricle. High-frame-rate velocity vector images of the cardiac muscle were generated with three common speckle-tracking approaches for subpixel estimation. Subpixel refinement based on differential optical flow was the most robust in the left ventricle of 10 subjects and allowed accurate high-frame-rate STE. This paper illustrates that coherent compounding with MoCo preserves the speckle patterns and makes it possible to carry out efficient speckle tracking. The 3-D speckle tracking of the myocardium will be the logical follow-up of the present findings.

Fig. 1 .

 1 Fig. 1. Examples of estimated in vitro velocity fields and the corresponding residuals (right column), when the disk outer speed was 15 cm/s. From top to bottom: 1) theoretical ground truth and velocity fields derived from: 2) correlation peak fitting; 3) phase correlation; and 4) optical flow.

Fig. 2 .

 2 Fig. 2. Examples of estimated velocity fields based on peak fitting, with and without MoCo. The disk outer speeds were (a) 2 and (b) 20 cm/s. In this example, the vectors were not smoothed. Different scales for the velocity vectors were used for a better rendering.

Fig. 3 .

 3 Fig. 3. In vitro normalized errors of the (a) radial and (b) cross-range velocities measured by peak fitting, phase correlation, and optical flow. The abscissa represents the outer speed of the disk.

Fig. 6 .

 6 Fig. 6. Distributions of the GLS peaks determined in 10 healthy volunteers by peak fitting, phase correlation, and optical flow. The GLS peak distribution returned by the GE clinical scanner is represented as a reference.

TABLE I ACQUISITION

 I AND POSTPROCESSING PARAMETERS FOR THE In Vitro AND In Vivo EXPERIMENTS WITH THE VERASONICS RESEARCH SCANNER

This work was supported in part by the Fonds de Recherche du Québec-Nature et Technologies, in part by the LABEX Center Lyonnais d'Acoustique, in part by ANR-10 et Simulation (PRIMES) under Grant ANR-10-LABX-0063, and in part by Investissements d'Avenir under Grant ANR-11-IDEX-0007. The work of H. Liebgott and P. Joos was supported by Région Rhône-Alpes under Grant Explora'Pro and Explora'Doc. The work of D. Garcia was supported by the Natural Sciences and Engineering Research Council of Canada under Grant RGPAS-477914-2015 and Grant RGPIN-04217-2015. The work of D. Garcia and G. Cloutier was supported by the Fonds de Recherche du Québec-Nature et Technologies under Grant 2016-PR-189822. The work of M. Baudet was supported by the French Federation of Cardiology.

Philippe Joos, photograph and biography not available at the time of publication.

Jonathan Porée, photograph and biography not available at the time of publication.

Hervé Liebgott, photograph and biography not available at the time of publication.

Didier Vray, photograph and biography not available at the time of publication.

Mathilde Baudet, photograph and biography not available at the time of publication.

Julia Faurie, photograph and biography not available at the time of publication.

François Tournoux, photograph and biography not available at the time of publication.

Guy Cloutier, photograph and biography not available at the time of publication.

Barbara Nicolas, photograph and biography not available at the time of publication.

Damien Garcia, photograph and biography not available at the time of publication.