Nan Li

Peixun Fan
email: fanpeixun@gmail.com

Qiuchi Zhu

Bai Cui

Jean-Francois Silvain

Yong Feng Lu

Femtosecond laser polishing of additively manufactured parts at grazing incidence

Keywords: Additive manufacturing, Femtosecond laser, Laser polishing, Grazing incidence

Surface polishing is usually a requisite for making additively manufactured (AM) parts ready for practical applications. Due to its advantages of being flexible and noncontact, laser polishing has attracted increasing research interest. In this research, femtosecond (fs) laser polishing was established to post-process both the top surfaces and sidewalls of AM parts. The challenge to remove three levels of roughness (i.e., the initial surface roughness of the AM parts, the undulations newly introduced during fs laser polishing, and the micro-nanoscale surface features induced by fs laser irradiation) was identified and addressed. Mirror surfaces with Sa < 200 nm were achieved on stainless steel parts printed with an initial roughness > 20 µm. Both parallel-and perpendicular-incidence were investigated for the polishing, with the former verified to be more effective in eliminating the initial roughness of the AM parts, due to the elongated focal intensity profile of a Gaussian beam irradiated on the AM part surfaces. The challenge of forming threezone surfaces during the parallel-incidence was further addressed through a grazing-incidence polishing approach, and uniform smooth surfaces were realized. Fine-tuning the laser power enabled controlling the submicron surface features formed under fs laser irradiation, which determined the final achievable surface roughness.

Introduction

Nowadays, additive manufacturing (AM) has been increasingly applied in various fields including automobile, aerospace, biomedicine, etc., providing high flexibility in producing complex parts with desired performances [START_REF] Ngo | Additive manufacturing (3D printing): A review of materials, methods, applications and challenges[END_REF][START_REF] Blakey-Milner | Metal additive manufacturing in aerospace: A review[END_REF][START_REF] Wang | Applications of additive manufacturing (AM) in sustainable energy generation and battle against COVID-19 pandemic: The knowledge evolution of 3D printing[END_REF][START_REF] Yin | Design and characterization of radar absorbing structure based on gradient-refractive-index metamaterials[END_REF][START_REF] Najmon | Review of additive manufacturing technologies and applications in the aerospace industry[END_REF][START_REF] Popov | Design and 3D-printing of titanium bone implants: brief review of approach and clinical cases[END_REF][START_REF] Liu | 3D Printing Surgical Implants at the clinic: A Experimental Study on Anterior Cruciate Ligament Reconstruction[END_REF]. Post-AM processing, polishing in particular, is usually necessary for improving the surface qualities of AM parts due to their intrinsic rough surfaces (typically Ra > 15 µm) resulting from the line-by-line and layer-by-layer printing process and the insufficient melting of the printing powders on surfaces [START_REF] Ali | Identification and characterization of spatter particles and their effect on surface roughness, density and mechanical response of 17-4 PH stainless steel laser powder-bed fusion parts[END_REF][START_REF] Guo | Effect of processing parameters on surface roughness, porosity and cracking of as-built IN738LC parts fabricated by laser powder bed fusion[END_REF]. Among different polishing techniques, laser polishing has attracted increasing research interest for achieving high-quality surface finishes due to its advantages of being flexible, noncontact, and less material dependence.

Laser polishing primarily uses continuous-wave (CW) or long-pulse [above nanosecond (ns)] lasers to melt thin surface layers, which promotes the re-flow or re-distribution of surface materials to reduce surface roughness [START_REF] Zhang | Implications of using two low-power continuous-wave lasers for polishing[END_REF][START_REF] Temmler | Influence of pulse duration and pulse frequency on micro-roughness for laser micro polishing (LµP) of stainless steel AISI 410[END_REF]. For AM parts, surface polishing of titanium alloy [START_REF] Tian | Material interactions in laser polishing powder bed additive manufactured Ti6Al4V components[END_REF][START_REF] Ma | Laser polishing of additive manufactured Ti alloys[END_REF][START_REF] Marimuthu | Laser polishing of selective laser melted components[END_REF][START_REF] Guan | Laser polishing of additive-manufactured Ti alloys and Ni alloys[END_REF], nickel alloy [START_REF] Guan | Laser polishing of additive-manufactured Ti alloys and Ni alloys[END_REF], and stainless steel [START_REF] Bhaduri | Laser polishing of 3D printed mesoscale components[END_REF][START_REF] Chen | Modification of surface characteristics and electrochemical corrosion behavior of laser powder bed fused stainlesssteel 316L after laser polishing[END_REF][START_REF] Lamikiz | Laser polishing of parts built up by selective laser sintering[END_REF] has been realized using ns and CW lasers. However, laser polishing through thermal melting is usually dependent on the initial surface roughness of AM parts. Surfaces with low initial roughness (e.g., 1-8 µm) can be smoothed down to nanoscale roughness [START_REF] Tian | Material interactions in laser polishing powder bed additive manufactured Ti6Al4V components[END_REF][START_REF] Bhaduri | Laser polishing of 3D printed mesoscale components[END_REF][START_REF] Zhihao | Laser polishing of additive manufactured superalloy[END_REF][START_REF] Santos Solheid | Laser surface modification and polishing of additive manufactured metallic parts[END_REF], whereas surfaces with high initial roughness (e.g., 8-25 µm) can hardly be smoothed to roughness below 2 µm [START_REF] Genna | Laser finishing of Ti6Al4V additive manufactured parts by electron beam melting[END_REF][START_REF] Liang | Laser polishing of Ti6Al4V fabricated by selective laser melting[END_REF][START_REF] Zhang | Investigation of laser polishing of four selective laser melting alloy samples[END_REF][START_REF] Bobe | Experimental study and empirical modelling of laser surface finishing of silicon carbide[END_REF]. In addition, critical applications (e.g., optical, electrical, and biomedical devices) usually have strict requirements on surface microstructures, compositions, and properties, ruling out the thermal-melting based methods which may cause surface microstructural and compositional changes or induce residual stresses and surface distortions [START_REF] Zhao | Formation mechanism of a smooth, defect-free surface of fused silica optics using rapid CO2 laser polishing[END_REF]. It is also challenging to polish materials sensitive to heat or with high melting points using thermal melting processes [START_REF] Zak | Additive manufacturing of polymer-derived ceramics[END_REF][START_REF] Zhang | 3D printing of transparent YAG ceramics using copolymerassisted slurry[END_REF][START_REF] Carloni | Transparent alumina ceramics fabricated by 3D printing and vacuum sintering[END_REF][START_REF] Dizon | Mechanical characterization of 3Dprinted polymers[END_REF][START_REF] Zhang | Three-dimensional printing of transparent ceramics by lithography-based digital projection[END_REF][START_REF] Li | 3D printing to innovate biopolymer materials for demanding applications: A review[END_REF].

In the past decade, femtosecond (fs) lasers have been recognized as a powerful tool for precision machining and micro/nanofabrication [START_REF] Zhu | Femtosecondlaser sharp shaping of millimeter-scale geometries with vertical sidewalls[END_REF][START_REF] Fan | General strategy toward dual-scale-controlled metallic micro-nano hybrid structures with ultralow reflectance[END_REF][START_REF] Zhang | Bioinspired micro/nanostructured surfaces prepared by femtosecond laser direct writing for multifunctional applications[END_REF][START_REF] Liu | Precise assembly and joining of silver nanowires in three dimensions for highly conductive composite structures[END_REF] due to their advantages over CW and longpulse lasers, including having less thermal effect and minimal influence on the initial surface compositions. Different from the CW and long-pulse laser polishing, which is mainly an equalmaterial manufacturing process, fs laser processing mainly utilizes a subtractive ablation process that can not only reduce the surface roughness but also provides a second chance to improve the dimensional accuracies of three-dimensional (3D) parts. The fs laser polishing could be used either as an alternative to or in combination with CW and long-pulse laser polishing to achieve the lowest surface roughness in a more efficient manner. Due to its promising potential, a few attempts have been made in using fs laser ablation to polish planar surfaces, mainly with initially low roughness [START_REF] Taylor | Femtosecond laser polishing of germanium[END_REF][START_REF] Yang | Influence of energy fluence and overlapping rate of femtosecond laser on surface roughness of Ti-6Al-4V[END_REF][START_REF] Chang | Surface polishes of the SKD 61 tool steel by a femto pulse laser operating in a wide range of powers[END_REF]. Nevertheless, fs laser polishing of 3D AM parts is yet to be fully explored.

In this study, fs laser polishing was established to post-process both the top surfaces and sidewalls of AM parts. The advantage of parallel-incidence over perpendicular-incidence polishing in realizing lower roughness was identified and ascribed to different laser-matter interactions occurring at different angles of incidence (AOI). Further, to address the challenge of forming threezone surfaces during parallel incidence (AOI = 90°), a grazing-incidence (i.e., with grazing angles (GA) of 1-5°) was used, which improved the surface finish of the AM parts over 100 times. Surface roughness down to < 200 nm was uniformly achieved on the surfaces with initial roughness > 20 µm (Fig. 1). The evolution of submicron surface features under fs laser polishing were also investigated. Lower surface roughness was achieved when the dominating surface features changed from continuous ripples to random, finer particles. This research has laid a foundation to make fs laser polishing an effective technique for post-processing AM parts.

Materials and methods

As a widely used engineering material, SAE 316L stainless steel was selected as a typical example in this research. The specimens were selectively laser-melted blocks of 10×10×3 mm 3 with an initial surface roughness of Sa = 15-28 µm, which were obtained from Proto Labs, Inc.

The laser polishing was conducted in open atmosphere using a Tangor laser system which works at a wavelength of 1030 nm and tunable pulse durations of 400 fs -10 ps (Amplitude Laser, Inc.).

The p-polarized laser beam was scanned by a galvanometer scanner (SCANLAB GmbH) and focused to a spot of about 30 µm in diameter by an F-theta lens with a focal length of 70 mm. The scanning field of the galvanometer scanner is ~30×30 mm 2 . The AOI was controlled by a GN1 goniometer (Thorlabs) which is a device capable of rotating or tilting an object precisely, within a small angular range. The specimens together with the goniometer were fixed on an X-Y-Z stage which can reach travel ranges of 300×300×50 mm 3 . The blocks of 10×10×3 mm 3 in size were used only for demonstration of the fs laser polishing approach. Dimensions of AM parts that can be polished are determined by the scanner and stages used. Surface roughness and 3D morphology images were measured and collected using either a VK-X200K laser scanning microscope (LSM, Keyence) or a NewView 8000 3D optical surface profiler (Zygo). The microstructures on the polished surfaces were characterized using an FEI Helios Nanolab 660 dual-beam field-emission scanning electron microscope (SEM) which was equipped with an energy dispersive X-ray spectroscopy (EDS) detector. X-ray diffraction (XRD) analyses were performed on a Bruker-AXS D8 diffractometer.

A three-step polishing approach was developed (i.e., sequentially by coarse polishing, precision polishing, and finishing steps) for the parallel-and grazing-incidence polishing, as schematically shown in Fig. S1. In each step, the fs laser scanning was continuously performed via the galvanometer scanner while continuously lowering the focal plane through a Z-stage until the entire depth was polished. Subsequently, the focal plane was moved back to the top surface to conduct the next step. The specific processing parameters for each step were listed in Table 1.

Herein, scanning times refer to the number of laser scans that were evenly made within every 1 mm-depth range. The processing parameters in Table 1 were optimized in our preliminary experiments. The main difference between the coarse and precision polishing steps was that higher pulse energies were used in the coarse polishing to produce a relatively flat base for the subsequent precision polishing, while a higher pulse repetition rate was used in the precision polishing to realize a more uniform surface. Surface material layers of 60 and 20 µm thicknesses were removed through the coarse and precision polishing steps, respectively. The final finishing step was used to further modify and smooth the surfaces from the precision polishing step, without feeding the fs laser to remove more surface materials. Although few deep cavities may not be completely removed, most surface areas of the AM specimens used in this research can be smoothened by removing 80 µm surface layers. For AM parts having rougher initial surfaces, the complete removal of all surface defects can be realized by simply increasing the thickness of surface layers to be removed in the coarse polishing step. Such a three-step approach can be used to realize both lower surface roughness and higher processing efficiency.

Results and discussion

Top surface and sidewall polishing of three-level roughness

The initial top surfaces and sidewalls of the as-printed samples via selective laser melting were obviously rough and occupied by incompletely melted printing powders, laser printing tracks, and random surface defects (e.g., cracks and voids) (Fig. 1a). Bumps were also observed at the edges between the top surface and sidewalls (Fig. 1b). After fs laser polishing, both the top surface and sidewalls were remarkably smoothed. The edges between the top surface and sidewalls were also sharpened (Fig. 1d,e). The polished surfaces became mirror-like surfaces that can show reflections of objects (e.g., a tweezer) near them (Fig. 1d), indicating the effectiveness of the fs laser as a tool for reducing surface roughness. s

Specific parameters Coarse polishing Precision polishing Finishing

Laser power Optimized in Figs. 5,6 Optimized in Fig. 7 Optimized in Fig. S6 Pulse duration (fs) 400 400 400 fs -10 ps Repetition rate (kHz) 66 99 99

Scanning speed (m/s) 1 1 1

Scanning times per mm 1000 1000 1000 Specifically, three levels of roughness need to be removed to achieve mirror-like surfaces: (ⅰ) the initial surface roughness of selective laser melted parts (Fig. 2a), including the laser printing tracks and incompletely melted powders, etc., (ⅰⅰ) the undulations introduced by fs laser scanning during the polishing process, and (ⅰⅰⅰ) the micro-nanoscale surface features induced by fs laser irradiation. Therefore, the key to fs laser polishing AM parts is to minimize the initial roughness while preventing the formation of new undulations and micro-nanoscale features. Two different ways in which the fs laser was incident on the AM parts were investigated, i.e., the perpendicular incidence (AOI = 0°, or top-surface polishing, Fig. 2b) and the parallel incidence (AOI = 90°, or sidewall polishing, Fig. 2e). Various conditions were investigated for the perpendicular-incidence polishing (Figs. S2 andS3). However, it was found that the fs laser polishing at perpendicular incidence could not sufficiently reduce the initial roughness (Figs. 2c,d). For example, shallower laser printing tracks and deep cavities can still be observed on the surfaces, covered with some micro-nanoscale features generated by fs laser irradiation (Figs. 2d andS3). In contrast, the fs laser polishing at the parallel incidence can eliminate the initial roughness, leaving only slight undulations from fs laser scanning and micro-nanoscale features induced by fs laser irradiation (Figs. 2f,g).

The advantage of the parallel-incidence over perpendicular-incidence polishing is ascribed to the elongated focal intensity profile of a Gaussian beam. Usually, the depth of focus (Fd, defined as twice of the Rayleigh length) of a Gaussian laser beam is much larger than its beam waist. The depth of focus is determined by the laser wavelength (λ) and beam waist radius (w0) as,

𝐹 𝑑 = 2𝜋𝑤 0 2 𝜆 . (1)
Calculated from Eq. ( 1), the depth of focus of the fs laser used in this research was 1372 µm, which was over 45 times of the beam waist (i.e., 30 µm used in this study). As a result, thin material layers were simultaneously removed from both the peak and valley areas at perpendicular incidence, creating new peaks and valleys (Fig. S4b). Therefore, the laser printing tracks from the selective laser melting process could be reduced but not be sufficiently removed. At parallel incidence, the materials at different heights were not simultaneously ablated but successively removed due to the small beam waist. Therefore, the peak areas were preferentially ablated layer by layer (Fig. S4c), which significantly reduced the initial surface roughness of AM parts. process, schematic of effect, and a typical Zygo image for the parallel-incidence polishing, respectively (using the parameters in Table 1 with a laser power of 9.4, 11, and 11 W for the coarse polishing, precision polishing, and finishing steps, respectively).

Grazing-incidence fs laser polishing

Despite its advantages in achieving smoother surfaces, the fs laser polishing at parallel incidence (AOI = 90°) encountered a challenge of forming three-zone surfaces in the experiments.

Three zones were observed on the fs laser polished areas: the top rough zone, the middle smooth zone, and the bottom rough zone (Fig. 3a,b). The as-printed zone to be polished was below the bottom rough zone. Both the top and bottom rough zones resulted from fs laser ablation under outof-focus conditions (Fig. 3b). The top zone was initially smoothed when the fs laser was focused there. As the fs laser polishing went deeper with the top zone being re-ablated under out-of-focus conditions, the top zone became rough again. The bottom zone was always ablated by the fs laser under out-of-focus conditions and, hence, was insufficiently polished.

As evidenced in Fig. 3c, focusing the fs laser on the top of the sidewall (e.g., depth (Z) = 0.25 mm) initially produced a narrow smooth zone there (the smooth zone) and a wide rough zone below (i.e., the bottom rough zone). As the fs laser polishing went deeper (e.g., Z = 0.5 mm), the bottom rough zone also moved down, and the smooth zone was widened. However, when the fs laser polishing went further deeper (e.g., Z = 0.7 mm), it damaged the top part of the smooth zone and formed a top rough zone. As the fs laser polishing went even deeper (e.g., Z = 1 mm), the top rough zone was widened to ~260 µm. Two clear boundaries between the three zones can be observed, indicating the evolution of these three zones. Since the bottom rough zone can be eventually smoothed as the fs laser polishing continuously goes deeper, the key challenge of overcoming the three-zone surfaces and obtaining uniform smooth surfaces is to remove the top rough zone.

The formation of the top rough zone under out-of-focus conditions was caused by the divergence of the fs laser beam. In the experiment, the diameter (D) of the fs laser beam entering the galvanometer scanner was ~10 mm and the focal length (f) of the F-theta lens was 70 mm.

Therefore, the half divergence angle can be estimated by

𝜃 = arctan( 𝐷 2𝑓 ), (2)
which was ~4.1°. To prevent the formation of the top rough zone, the AOI used for fs laser polishing needs to be adjusted accordingly. Since the divergence angle of the laser beam is small, only small GAs (e.g., 0-5°) were tested, through which a grazing-incidence fs laser polishing approach was developed (Fig. 4a,b). As shown in Fig. 4c, a wide top rough zone was formed at GA = 0°. As larger GAs were used, the width of the top rough zone was gradually reduced until it was too small to measure (Fig. 4c,d). A GA above 4° successfully prevented the re-ablation of the top zone, which is consistent with the half divergence angle estimated through Eq. (2). At the same time, the surface roughness stayed at low levels below 1 µm (Fig. 4e), confirming the capability of the grazing-incidence fs laser polishing in achieving uniform and smooth surfaces. Both the smooth top surface and sidewalls of the AM part in Fig. 1d were achieved through the grazing-incidence fs laser polishing. The sidewalls were firstly polished one by one. Then, the AM part was flipped by 90° to have the initial top surface stand vertically where the grazing-incidence fs laser polishing was performed, as shown in Fig. S5.

Optimization of grazing-incidence fs laser polishing for minimizing surface roughness

A systematic investigation was performed to identify the evolution of the surface roughness of AM parts with the surface dominating features during the grazing-incidence fs laser polishing, clarifying how the surface roughness was gradually reduced in three levels by adjusting the fs laser polishing process. As shown in Fig. 5, the fs laser reduces the initial surface roughness primarily through a layer-by-layer ablation mechanism. As thicker material layers were removed, the surfaces became smoother. Even when it was programmed to remove material layers of a same thickness (e.g., 60 µm), the obtained surface qualities varied with the specific processing parameters used. As an example, Fig. 6 shows the influences of the two main parameters (i.e., the laser power and the scanning time)

on the surface roughness of the AM parts. Briefly, when higher laser power and more scanning times were applied, the initial roughness (Level (ⅰ)) including the laser printing tracks and incompletely melted powders became blurry, as indicated by the arrow from the bottom-left to the top-right corners in Fig. 6. Specifically,  When less laser scans were applied within a unit depth range (e.g., scanning times = 250), the initial roughness from selective laser melting remained on the surfaces. As the laser power was increased from 7.9 to 11.0 W, the laser printing tracks and incompletely melted powders were reduced, and the surfaces became less bumpy.

 Using more laser scans within a unit depth range (e.g., scanning times = 500) significantly reduced the initial roughness at all laser powers between 7.9-11.0 W. Additionally, the laser printing tracks were flattened and the undulations introduced by the fs laser polishing (much finer than the laser printing tracks) were observed. At this stage, the undulations introduced by the fs laser scanning during the polishing process (Level (ⅰⅰ)) dominated the surfaces and accounted for the remaining surface roughness.

 When the number of laser scans applied within a unit depth range was further increased (e.g., scanning times = 1000), the undulations resulting from the fs laser polishing were reduced and even became unobservable. The dominating features on the surfaces of the AM parts became the submicron features induced by the fs laser (e.g., nanoripples and nanoparticles), achieving a surface roughness of the magnitude of ~100 nm.

The observations described above indicate that a careful control over the fs laser processing conditions (i.e., to maximumly reduce the initial roughness while suppressing the undulations and submicron features generated by fs laser simultaneously) is necessary for an effective fs laser polishing.

Fig. 6. A systematic investigation on the evolution of surface roughness with surface dominating features. Only the coarse polishing step was applied here.

The formation of submicron features on solid surfaces under fs laser irradiation has been extensively studied [START_REF] Gräf | Polarisation-dependent generation of fs-laser induced periodic surface structures[END_REF][START_REF] Huang | Origin of laser-induced near-subwavelength ripples: interference between surface plasmons and incident laser[END_REF]. The dependence of the achievable surface roughness on such submicron features distinguishes fs laser polishing from CW and long-pulse laser polishing. To investigate how fs-laser-induced submicron features evolve during the polishing process, rather than during the fs laser irradiation of smooth solid surfaces as in previous studies, the fs laserpolished surfaces were carefully examined through SEM.

Three types of submicron features were observed as the fs laser polishing conditions vary.

Taking laser power as an example (Fig. 7a-c), the submicron features evolved from continuous ripples to discontinuous ripples (ripples together with particles) and then to finer particles as the laser power was increased from 4.6 to 7.3 and then to 11.0 W. As a result, the surface roughness was reduced from above 1 µm to only 400 nm. The discontinuity of the ripples (i.e., the transition from ripples to particles) at a higher laser power was caused by more intense laser ablation with slight local melting. Further increasing the laser power to 11.0 W not only turned most of the submicron features on the polished surfaces to particles, but also reduced the size of these particles, which minimized the surface roughness. Furthermore, a fast-Fourier-transformation (FFT)

analysis was conducted on the SEM images. Bright spots can be observed in the FFT images at lower laser powers but became blurry at higher laser powers, confirming that the surface features evolved from more regular to more random ones, which are favorable for achieving lower surface roughness.

Specifically, the characteristics of submicron ripples can be defined by their vertical heights and horizontal periodicities (d). The formation of both low-spatial-frequency and high-spatialfrequency ripples on various solid surfaces under fs laser irradiation have been extensively studied [START_REF] Bonse | Femtosecond laser-induced periodic surface structures[END_REF]. In our experiments, the low-spatial-frequency ripples were found to dominate the surfaces.

Previous studies have correlated the periodicities of low-spatial-frequency ripples induced by ppolarized fs laser to laser wavelengths and incident angles as [START_REF] Bonse | Laser-induced periodic surface structures-a scientific evergreen[END_REF],

𝑑 = 𝜆 1±sin(AOI) . (3)
At a GA of 4°, the AOI is 86°. Hence, the periodicity of the fs-laser-induced ripples is 515.6 nm, as calculated from Eq. ( 3). As shown in Fig. 7a, the periodicity of the ripples formed during the grazing-incidence fs laser polishing is ~500 nm, which is consistent with the calculation. However, few investigations have been made on the heights of the submicron ripples, although they can influence more on the surface roughness. As a simple estimation, the heights of the fs-laserinduced submicron ripples are usually of the same magnitudes as their periodicities, which also implies a range of surface roughness of 100-1000 nm. To explore the potential to further smooth the fs-laser-induced submicron features and, hence, to reach lower surface roughness, a finishing step was conducted after completing both the coarse and precision polishing steps. Different pulse durations, pulse repetition rates, and laser powers were investigated for the finishing step (Fig. S6). Lower roughness values were achieved at the pulse duration of 400 fs compared with 1 and 10 ps. Surface roughness values even below 200 nm were measured after the fs laser finishing step, where micro-melted surface regions were observed (Fig. 7d). Further investigations should focus on controlling and reducing the submicron features to achieve surface roughness below 100 nm using fs lasers. It is known that both the fs laser ablation/melting thresholds and the sizes of submicron features formed under fs laser irradiation vary among different materials. Therefore, we believe it is possible to realize even lower surface roughness on some other materials using the fs laser polishing approach established in this study.

Besides, it was recently reported that continuous surface melting can even be realized by fs lasers (or ps lasers in more studies) through heat accumulation via MHz-level pulse repetition rates [START_REF] Hafiz | Experimental analysis of applicability of a picosecond laser for micro-polishing of micromilled Inconel 718 superalloy[END_REF][START_REF] Chen | Picosecond laser pulse polishing of ASP23 steel[END_REF][START_REF] Osbild | Ultrashort pulse laser micro polishing of steel -Investigation of the melt pool depth[END_REF][START_REF] Sassmannshausen | Ultrashort pulse laser polishing by continuous surface melting[END_REF]. Such a fs-laser-induced continuous surface melting process could also benefit the achievement of extremely low surface roughness for AM parts.

Advantages of fs laser polishing

The surface compositions of the AM parts after the fs laser polishing were examined through The EDS analyses further confirmed that the main element contents (Fe, Cr, Ni, Mo) were not obviously changed during the fs laser polishing (Fig. 8b and Table 2). The rise of the oxygen content indicates the occurrence of slight surface oxidation, which did not influence the main crystalline microstructures of the AM parts, according to the XRD results.

It is well-known that fs lasers can process almost any solids with little thermal effects.

Therefore, chemical-composition and material-property changes usually seen in thermal treatments will be insignificant under fs laser irradiation. As for the influences of the fs-laserinduced submicron features on the surface physical-chemical properties, extensive understanding has been made through previous studies on various materials [START_REF] Fan | General strategy toward dual-scale-controlled metallic micro-nano hybrid structures with ultralow reflectance[END_REF][START_REF] Zhang | Bioinspired micro/nanostructured surfaces prepared by femtosecond laser direct writing for multifunctional applications[END_REF][START_REF] Vorobyev | Direct femtosecond laser surface nano/microstructuring and its applications[END_REF][START_REF] Stratakis | Laser engineering of biomimetic surfaces[END_REF], which can also apply to the submicron features achieved after the three-step fs laser polishing. As a comparison, representative results from previous studies on polishing AM parts with CW or long-pulse lasers were summarized in Fig. 9. It can be seen that the surface finish achieved through the fs laser polishing approach in this research is comparable to the best levels achieved by CW and long-pulse lasers. In addition, the effect of CW and long-pulse laser polishing shows an obvious dependence on the initial surface roughness. A final surface roughness of Sa ~ 200 nm was obtained through fs laser polishing the surface with an initial roughness of Sa = 25 µm, equivalent to > 99% improvement in the surface quality. It is also worth noting that although the experiments were implemented on 316L stainless steel in this research, the knowledge accumulated and approaches developed in this study can be widely applied to polishing AM parts of other materials (e.g., metals, ceramics, polymers, and composites). Nevertheless, although it can be used to polish surfaces with high initial roughness, the fs laser polishing is not dedicated to this purpose. The results obtained in this research can support customizable polishing from different initial roughness levels towards different targeted ones, such as processes X1-Z1 shown in Fig. 10. Practical post-AM processing can comprise a series of approaches including the mechanical and/or chemical ones. Rather than replacing any existing post-AM processing approaches, the fs laser polishing can be used in combination with any of them. Regardless of the specific roughness levels to which the AM parts are processed by other 20 approaches, fs laser polishing can further improve the surface finishes.

Besides, the cuboid blocks were used only as an example of 3D AM parts, and the top surface and sidewalls of the cuboid blocks were also investigated as representative surfaces of 3D AM parts. The concepts and fundamentals established in this research can still apply when polishing AM parts with complex shapes. Specifically, hardware configurations and system integration with more flexible motion systems (e.g., translation stages with longer travel ranges, motorized rotation and tilt stages, multi-axis scanners and stages, etc.) may be needed to facilitate the continuous control over the fs laser scanning along non-flat surfaces to conduct the grazing-incident polishing.

From the point of view of practical applications, the efficiency of the fs laser polishing was also estimated, which is ~30 min/cm 2 , limited by the current parameter set and hardware system.

Since the main concepts and fundamentals have been demonstrated in this research, the fs laser polishing approach can be flexibly extended to higher parameters (e.g., higher laser power, pulse repetition rate, and scanning speed) and upgraded hardware systems, which can significantly increase the processing efficiency.

Conclusions

In this study, fs laser polishing was established for post-processing both the top surfaces and sidewalls of AM parts. The challenge of removing three levels of roughness was addressed, achieving mirror-like surfaces with a Sa < 200 nm on stainless steel parts with initial roughness > 20 µm, equivalent to > 99% improvement on the surface finish. Parallel-incidence polishing was found to be more effective than perpendicular-incidence polishing in eliminating the initial roughness of the AM parts, which was ascribed to the elongated focal intensity profile of the Gaussian beam. Furthermore, the grazing-incidence fs laser polishing approach was developed to solve the issue of forming three-zone surfaces during the parallel incidence and produce uniform, smooth surfaces. The submicron surface features formed under fs laser irradiation determined the final achievable surface roughness. Fine-tuning the laser power enabled controlling the submicron surface features, where random and finer particles were more favorable for reaching lower surface roughness than continuous ripples. No obvious composition changes were induced during fs laser polishing, and the original material properties were well preserved. This research demonstrates the capability of fs lasers in surface polishing and opens a new avenue for post-processing AM parts.

Fig. 1 .

 1 Fig. 1. Fs laser polishing and the representative results: (a) photo and (b) LSM image of the as printed part; (c) schematic of the fs laser polishing approach; (d) photo and (e) LSM image of the fs laser polished part; a tweezer was positioned near the polished sidewall (d) and the top surface (inset in (d)) to show the mirror-like surfaces achieved.

Fig. 2 .

 2 Fig. 2. Comparison of the fs laser perpendicular-and parallel-incidence polishing: (a) a typical Zygo image of the as-printed surfaces; (b), (c), and (d) schematic of process, schematic of effect, and a typical Zygo image for the perpendicular-incidence polishing, respectively (processing parameters: laser power = 1 W, repetition rate = 99 kHz, scanning speed = 1 m/s, hatching distance = 5 µm, scanning times = 800); (e), (f), and (g) schematic of

Fig. 3 .

 3 Fig. 3. The challenge of forming three-zone surfaces during fs laser polishing at parallel incidence: (a) schematics of the three-zone surfaces from both side (left) and front (right) views; (b) 3D schematic of the formation of the three-zone surfaces; (c) LSM images showing the evolution of the three-zone surfaces as the fs laser polishing went deeper. Only the coarse polishing step was applied here.

Fig. 4 .

 4 Fig. 4. Grazing-incidence fs laser polishing for achieving uniform smooth surfaces: (a) schematics of GA used from the side view (left) and the uniform surfaces from the front view (right); (b) 3D schematic of the grazingincidence fs laser polishing; (c) SEM images showing the reduction of the top rough zone as larger GAs were used; evolution of the width of the top rough zone (d) and surface roughness (e) with the GA. Both the coarse and precision polishing steps were applied here.

Fig. 5 .

 5 Fig. 5. Gradual reduction of initial surface roughness of AM parts in the fs-laser coarse polishing step: (a)-(d) typical Zygo images of the surfaces polished after material layers of different thickness were removed; (e)-(h) corresponding schematics of the fs laser polishing via a layer-by-layer ablation mechanism. Processing parameters used: laser power = 9.4 W, repetition rate = 66 kHz, scanning speed = 1 m/s, GA = 4°, scanning times = 1000.

Fig. 7 .

 7 Fig. 7. Evolution of submicron surface features with different laser powers in fs laser polishing and their influence on the final surface roughness. (a-c) Both the coarse and precision polishing steps were applied. The power shown for (a-c) was used for the precision polishing, and the parameters for the coarse polishing were the same for (a-c). (d) The finishing step was conducted on (c).

  XRD and EDS analyses. The three major peaks at 2θ of 43.55°, 50.55°, and 74.7° in the XRD patterns (Fig. 8a) correspond to the (111), (200), and (220) planes of the austenite (γ-Fe) phase, respectively. The positions of the three XRD peaks of the polished surface remained the same as the initial rough surface, indicating that no significant phase change occurred.

Fig. 8 .

 8 Fig. 8. (a) XRD and (b) EDS analyses on the polished and as-printed surfaces.

Fig. 9 .

 9 Fig. 9. Comparison of the roughness reduction between this work and previous reports.

Fig. 10 .

 10 Fig. 10. Roadmap for customizable fs laser polishing approaches from different initial roughness levels towards different targeted ones.

Table 1 .

 1 Summary of the process parameters in fs laser polishing

Table 2 .

 2 Chemical compositions of both the as-printed and polished surfaces determined by EDS.

	Elements	Fe	Cr	Ni	Mo	C	O	Si
	As-printed	63.99	22.04	9.69	0.72	0.40	1.84	1.32
	wt.%
	Polished	62.61	21.34	10.31	0.64	0.53	4.34	0.22

Acknowledgement

This study was supported by the National Science Foundation (CMMI 1826392) and the Nebraska Center for Energy Sciences Research (NCESR). The research was performed in part in the Nebraska Nanoscale Facility: National Nanotechnology Coordinated Infrastructure and the Nebraska Center for Materials and Nanoscience, which are supported by the National Science Foundation under Award ECCS: 1542182, and the Nebraska Research Initiative.


Sana Jemel

Yannick Raveloarisaona

Anne-Laure Bidaud

Elie Djenontin

Aicha Kallel

Jacques Guillot
email: jacques.guillot@oniris-nantes.fr

Kalthoum Kallel
email: kallelkalthoum@gmail.com

Françoise Botterel
email: francoise.botterel@aphp.fr

Eric Dannaoui
email: eric.dannaoui@aphp.fr

In vitro and in vivo evaluation of antifungal combinations against azole-resistant Aspergillus fumigatus isolates

Keywords: Aspergillus fumigatus, antifungal combination, Galleria mellonella, voriconazole, posaconazole, caspofungin, azole-resistance

Azole resistance in Aspergillus fumigatus (Af) has become a widespread threat and a major concern for optimal management of patients with invasive aspergillosis (IA). Combination of echinocandins with azoles is an attractive alternative option for the treatment of IA due to azole-resistant Af strains. The aim of this study was to evaluate the in vitro and in vivo combination of caspofungin (CAS) with either voriconazole (VRZ) or posaconazole (PSZ). In vitro interactions were assessed by two methods, and an animal model of IA in Galleria mellonella was used for in vivo evaluation. Assessment of efficacy was based on larvae mortality. Groups of 10 larvae were infected by 3 clinical strains of Af (azole susceptible, AfS; PSZ resistant, AfR1; VRZ and PSZ resistant strain, AfR2). In vitro, combination of CAS and azoles was indifferent against AfS, and AfR2, and a synergy was found for AfR1. When compared to VRZ monotherapy, the combination of VRZ at 4 µg/larva with CAS at 4 µg/larva improved survival of AfR2-infected larvae (p=0.0066). Combination of PSZ at 4µg/larva with CAS at 4 µg/larva improved survival of AfR1-infected larvae compared to CAS (p=0.0002) and PSZ (0.0024) monotherapy. Antagonism was never observed. In conclusion, the combination of caspofungin with azoles is a promising alternative for the treatment of azole resistant strains of Af.

Introduction

Aspergillus fumigatus (Af) remains one of the most potent opportunistic fungal pathogens in humans. It causes a wide range of infections including invasive aspergillosis (IA), a severe condition occurring classically in immunocompromised patients. More recently, other risk factors of IA, such as severe influenza [START_REF] Schauwvlieghe | Invasive aspergillosis in patients admitted to the intensive care unit with severe influenza: a retrospective cohort study[END_REF][START_REF] Verweij | Review of influenza-associated pulmonary aspergillosis in ICU patients and proposal for a case definition: an expert opinion[END_REF] or severe COVID-19 [START_REF] Pasquier | A review of significance of Aspergillus detection in airways of ICU COVID-19 patients[END_REF][START_REF] Gangneux | Fungal infections in mechanically ventilated patients with COVID-19 during the first wave: the French multicentre MYCOVID study[END_REF] have been recognized. IA is associated with high mortality [START_REF] Latge | Aspergillus fumigatus and aspergillosis in 2019[END_REF][START_REF] Thompson | Aspergillus Infections[END_REF], despite effective first line treatment based on azoles [START_REF] Patterson | Practice guidelines for the diagnosis and management of aspergillosis: 2016 update by the Infectious Diseases Society of America[END_REF][START_REF] Ullmann | Diagnosis and management of Aspergillus diseases: executive summary of the 2017 ESCMID-ECMM-ERS guideline[END_REF]. Azoles are inhibitors of the sterol 14 α demethylase enzyme, a key step in ergosterol biosynthesis pathway. However, extensive use of azole drugs in the prevention and treatment of fungal infections, and extensive use of fungicides in agriculture, have contributed to the emergence of azole resistance in Af (Lestrade et al., 2019b). Different mechanisms of azole resistance have been reported [START_REF] Dudakova | Molecular tools for the detection and deduction of azole antifungal drug resistance phenotypes in Aspergillus species[END_REF]. The most important is related to changes in the target enzyme by mutation of its gene, but drug efflux has also been reported and resistance can be multifactorial [START_REF] Dudakova | Molecular tools for the detection and deduction of azole antifungal drug resistance phenotypes in Aspergillus species[END_REF][START_REF] Rybak | Emerging threat of triazole-resistant Aspergillus fumigatus[END_REF]. The emergence of azole resistance in Af makes the management of invasive aspergillosis more complex [START_REF] Verweij | International expert opinion on the management of infection caused by azole-resistant Aspergillus fumigatus[END_REF]. Azole resistance has been associated with treatment failure and excess mortality (Lestrade et al., 2019a;[START_REF] Resendiz-Sharpe | Prevalence of voriconazole-resistant invasive aspergillosis and its impact on mortality in haematology patients[END_REF].

Therefore, development of alternative treatment options for IA is necessary. The combination of an azole with an echinocandin is one of the therapeutic options (Verweij et al., 2015;[START_REF] Ullmann | Diagnosis and management of Aspergillus diseases: executive summary of the 2017 ESCMID-ECMM-ERS guideline[END_REF]. The complete evaluation of the efficacy of this kind of combination is an essential step for the validation of the treatment. The aim of this study was to evaluate the in vitro and in vivo activity of caspofungin (CAS) in combination with voriconazole (VRZ) or posaconazole (PSZ). For in vivo evaluation, we used the Galleria mellonella model that has proven its contribution to the evaluation of antifungal efficacy for the treatment of IA [START_REF] Forastiero | In vivo efficacy of voriconazole and posaconazole therapy in a novel invertebrate model of Aspergillus fumigatus infection[END_REF][START_REF] Maurer | Galleria mellonella as a host model to study Aspergillus terreus virulence and amphotericin B resistance[END_REF][START_REF] Jemel | Galleria mellonella for the evaluation of antifungal efficacy against medically important fungi, a narrative review[END_REF][START_REF] Jemel | In vivo efficacy of voriconazole in a Galleria mellonella model of invasive infection due to azole-susceptible or resistant Aspergillus fumigatus isolates[END_REF].

Materials and Methods

Strains, medium and growth conditions

Three clinical strains of Af, isolated from respiratory samples, were used in the present study. Identification was confirmed by sequencing part of the gene encoding beta-tubulin.

The CYP51A gene and its promoter had been previously sequenced to determine the mutations involved in azole-resistance [START_REF] Jemel | In vivo efficacy of voriconazole in a Galleria mellonella model of invasive infection due to azole-susceptible or resistant Aspergillus fumigatus isolates[END_REF]. We included one azolesusceptible strain (AfS) with a wild type CYP51A sequence, one strain (AfR1) with a G54W mutation and one strain (AfR2) with a L98H point mutation in CYP51A in combination with a 34-bp tandem repeat in the promoter (TR34/L98H).

Subcultures were performed on Sabouraud dextrose agar (VWR, Fontenay-sous-bois, France) with chloramphenicol (Sigma-Aldrich, Saint Quentin-Fallavier, France). They were incubated for 7 days at 37°C to obtain sufficient sporulation.

In vitro susceptibility and interaction between caspofungin and azoles

Antifungal susceptibility testing was performed by two methods: the reference microdilution broth technique following the recommendations of the Antifungal Susceptibility Testing Subcommittee of the European Committee on Antimicrobial Susceptibility Testing (EUCAST-AFST), and a concentration gradient strip commercial method (Etest®). EUCAST was performed as recommended [START_REF] Arendrup | Method for the determination of broth dilution minimum inhibitory concentrations of antifungal agents for conidia forming moulds. EUCAST definitive document E[END_REF]. For azoles and amphotericin B, minimum inhibitory concentration (MIC) values were determined after 48h of incubation by using a complete inhibition endpoint. For CAS, minimal effective concentration (MEC) endpoints were determined. Gradient concentration strip method (Etest®, Biomérieux, Marcy-l'Etoile, France) was performed according to the manufacturer instructions and MICs were read after 48h of incubation.

In vitro activity of the combination of CAS with either VRZ or PSZ was first evaluated by the EUCAST reference method modified for a broth microdilution checkerboard procedure [START_REF] Vitale | Antifungal combinations[END_REF]Bidaud et al., 2021). Final concentrations ranged from 0.008 to 0.5 µg/mL for CAS, 0.008 to 4 µg/mL for VRZ and PSZ. The final inoculum size in the plates was 1-2.5x10 5 (CFU) mL -1 . Microplates were incubated at 37°C and read after 48h of incubation. A growth inhibition endpoint of 50% was used both for the drugs tested alone and in combination. The experiments were performed in triplicate in each of two independent experiments. Data were first analyzed by calculation of the fractional inhibitory concentration index (FICI) interpreted as follow: synergy for FICI ≤ 0.5, no interaction for FICI between 0.5 and 4, and antagonism for FICI > 4 [START_REF] Odds | Synergy, antagonism, and what the chequerboard puts between them[END_REF]. A Bliss independence-based method was also used as previously described [START_REF] Meletiadis | Assessing in vitro combinations of antifungal drugs against yeasts and filamentous fungi: comparison of different drug interaction models[END_REF]. Two parameters were calculated: the sum (ƩSSI) and the mean (MSSI) of percentages for all statistically significant interactions. Synergy was defined by a ƩSSI >200% and/or a 95% confidence interval of MSSI that did not include 0.

Activity of the combinations was also evaluated by a gradient concentration strip method (Etest®) as described previously [START_REF] Vitale | Antifungal combinations[END_REF]Bidaud et al., 2021). Briefly, after inoculation of RPMI plates, one strip of VRZ or PSZ were placed on the agar surface for one hour, removed, and a strip CAS was applied exactly on the same position. MICs of the drugs alone and in combination were read after 48h of incubation.

Galleria mellonella inoculation and treatment

Galleria mellonella infection

Larvae of G. mellonella (Kreca® Ento-Feed BV, Ermelo, Netherlands) were used throughout the experiments. In each set of experiments, larvae were randomly distributed in groups of 10 animals.

After culture of the three Af strains, the inoculum was prepared in phosphate-buffered saline containing 0.01% of Tween 20 (PBST), and spore suspensions were adjusted to the required concentration by counting conidia in a hemocytometer. Lethal doses 90% (LD 90 ) of each Af strain were previously determined [START_REF] Jemel | In vivo efficacy of voriconazole in a Galleria mellonella model of invasive infection due to azole-susceptible or resistant Aspergillus fumigatus isolates[END_REF]. The injection was carried out with 10 µL in the ventral side of the last proleg by using a Hamilton® syringe.

Drug preparation

For treatment experiments, VRZ (Vfend® [Pfizer]) and PSZ (Noxafil® [MSD]) were dissolved in 9‰ saline to obtain a stock solution at 10 mg/mL and 18 mg/mL, respectively. Required dosage was obtained by further dilutions in 9‰ saline. For CAS (Cancidas® [MSD]), powder was dissolved in 10 mL of sterile distilled water to obtain a stock solution at 5 mg/mL and further dilutions were performed in 9‰ saline.

Caspofungin and posaconazole monotherapy

Groups of 10 larvae were infected by DL 90 of each Af strains. Two hours after infection, larvae were treated by injection in the ventral side of animal. CAS or PSZ were used at 1, 2, 4 and 8 µg/larva. Larval survival was monitored daily for 7 days. Two control groups were used, the first group consisted of infected larvae inoculated with 9‰ saline at 2h after infection and the second group (to assess toxicity) was only inoculated with the highest doses of CAS or PSZ (8 µg/larva). All experiments were performed two times and results were pooled for analysis.

Treatment combination of voriconazole or posaconazole and caspofungin

Solutions of VRZ or PSZ (0.8 µg/µL) and CAS at 0.2, 0.4 and 0.8 µg/µL was obtained by carrying out dilutions in 9‰ saline. At equal volume and before inoculation to larvae, the VRZ solution was mixed with each solution of different concentration of CAS to obtain a combination of VRZ at 0.4 µg/µL and CAS at 0.1, 0.2 or 0.4 µg/µL. After infection by the three strains of Af, a volume of 10 µL of each antifungal combination was injected in the haemocoel of larvae 2h after infection. Two control groups were used, the first group consisted of infected larvae inoculated with 9‰ saline at 2h after infection.

The second group (to assess toxicity) was only inoculated with the highest doses of combination (CAS at 4 µg/larva combined with VRZ or PSZ at 4 µg/larva). Three groups were treated by single VRZ, PSZ or CAS at 4 µg/larva to assess the contribution of combination compared to monotherapy. All experiments were performed three times and results were pooled for analysis.

Statistical analysis

Mortality curves were generated by Kaplan Meier method and compared by the log-rank test. All analyzes were performed using GraphPad Prism V.3.0 software for Windows (GraphPad Software, San Diego, USA). A value of p<0.05 was considered to be significant.

Results

In vitro antifungal susceptibility to antifungals

The in vitro antifungal susceptibility of Af strains determined by EUCAST and Gradient Concentration Strip (GCS) is presented in Table 1. Using EUCAST, AfS with a wild type CYP51A sequence was azole-susceptible. AfR1 with a G54W mutation was resistant to PSZ and itraconazole but susceptible to VRZ. AfR2 with a L98H point mutation in CYP51A gene in combination with a 34-bp tandem repeat in the promoter (TR34/L98H), was resistant to the three tested azoles. Results obtained by the Etest® method were within +/-2 log 2 dilutions comparable to EUCAST values (Table 1, Figure S1). For CAS, MEC for AfS, AfR1 and AfR2 was 0.25, 0.5 and 0.5 µg/mL, respectively. CAS MIC values determined by Etest® was systematically lower than EUCAST MEC values.

In vitro activity of antifungal combinations

When VRZ was combined with CAS, no interaction was observed between the two drugs by FICI (Table 2). The lowest FICI for the combination was 1.01, 0.75 and 1.25 for AfS, AfR1 and AfR2, respectively. Bliss analysis showed a synergistic interaction for AfR1 (ƩSSI >200% and 95% CI of MSSI did not include 0), but no interaction for AfS and AfR2 (Table 4). Antagonism was not detected for any of the strains.

When PSZ was combined with CAS, no interaction was observed between the two drugs by FICI (0.51) for AfS and AfR2 (Table 3). Due to the high level of PSZ resistance (high off-scale MIC), FICI was not computable for AfR1. By Bliss analysis, a synergistic interaction was observed for AfR1, but no interaction for AfS and AfR2 (Table 4). No antagonism was observed. Combinations were also evaluated by Etest® (Figure S2, Figure S3). Combinations were indifferent against all strains (Table S1, Table S2). There was no antagonism.

Evaluation of caspofungin monotherapy in Galleria mellonella

For control groups, without treatment, the mortality was at least 95% by day 7, with a median survival time of 3 days for AfS and AfR1 and 3.5 days for AfR2 (Figure 1). . In AfSinfected groups, CAS at 2, 4, and 8 µg/larva significantly increased the survival during the 7 days of experiment (p=0.0064, 0.017 and 0.0009, respectively). There was no difference in term of efficacy between the different doses of CAS. For AfR1-infected larvae, CAS did not provide any significant improvement in survival with a median survival time of 3 days. For AfR2-infected larvae, only CAS at 4 µg/larva significantly decreased the mortality when compared to the untreated control group (p=0.02).

Evaluation of posaconazole monotherapy in Galleria mellonella

For each strain, efficacy of PSZ at 1, 2, 4 and 8 µg/larva was evaluated (Figure 2). Mortality by day 7 in untreated larvae was 90%, 100% and 90% for AfS, AfR1 and AfR2, respectively.

Treatment at 4 µg/larva increased survival for AfS (p=0.0004) and AfR1 (p=<0.0001) but not for AfR2-infected larvae (p=0.41). In AfS-and AfR1-infected larvae the rate of survival was dose dependent. Although PSZ improved survival compared to untreated controls for both AfS and AfR1-infected larvae, the drug was more effective in AfS than in AfR1-infected larvae. Survival at day 7 was 10%, 50% and 70% for AfS-infected larvae while it was 0%, 20% and 40% for AfR1-infected larvae after PSZ treatment at 2, 4 and 8 µg/larva, respectively. Moreover, median survival for AfS infected larvae and treated with PSZ at 8 and 4 µg/larva was more than >7 days and 7 days compared to 2.5 and 3 days for AfR1-infected larvae.

Evaluation of combination of voriconazole with caspofungin in Galleria mellonella

For each isolate, larvae infected with LD 90 were treated with VRZ at 4 µg/larva combined with CAS at 1, 2 or 4 µg/larva at 2h post infection (Figure 3). Mortality, in untreated control groups was at least 95% at day 7 post infection. CAS monotherapy was effective only for AfSinfected larvae (p<0.0001). VRZ monotherapy significantly increased survival of AfS (p<0.0001), AfR1 (p<0.0001) and AfR2-infected groups (p=0.02) compared to untreated group. Nevertheless, the efficacy was better against AfS and AfR1 (survival of 35% and 30%, respectively) than against AfR2 (survival of 10%). The combination of VRZ (4 µg/larva) with CAS (4 µg/larva) significantly increased the survival of AfS (p<0.0001), AfR1 (p<0.0001) but not AfR2-infected larvae (p<0.25) compared to CAS monotherapy at 4 µg/larva. When compared to VRZ monotherapy, the combination (VRZ4 + CAS4) improved survival of AfR2infected larvae (p=0.0066), but not of larvae infected by AfS (p=0.24) or AfR1 (p=0.28). At a lower concentration, CAS at 1 and 2 µg/larva combined with VRZ at 4 µg/larva did not increase the survival for any of the strain, when compared to VRZ monotherapy.

Evaluation of combination of posaconazole with caspofungin in Galleria mellonella

For each Af strain, larvae were infected by LD 90 and treated after 2h by PSZ at 4 µg/larva monotherapy or combined with CAS at 1, 2 or 4 µg/larva (Figure 4). At day 7 post-infection, the mortality in the untreated control groups was >90%. Treatment by CAS at 4 µg/larva significantly improved survival for AfS (p<0.0001), but not for AfR1 (p=0.02) or AfR2-infected larvae (p=0.07) compared to untreated group. PSZ at 4 µg/larva significantly improved survival only for AfS (p<0.0001) and AfR2 (p=0.0018) but not for AfR1-infected larvae (p=0.78) compared to the untreated controls. Combination of PSZ at 4 µg/larva and CAS at 4 µg/larva improved survival only for AfR1-infected larvae compared to CAS (p=0.0002) and PSZ (p=0.0024) monotherapy.

Discussion

In the present study we found very weak in vitro interactions between caspofungin and azoles by checkerboard and by agar diffusion. A synergistic interaction was only found for one of the resistant strains (AfR1 resistant to ITZ and PSZ, susceptible to VRZ) when data were analyzed by a Bliss independence-based mathematical model. It has to be noticed that, although in vitro testing of antifungal combinations against filamentous fungi are very useful, the techniques are not well standardized, and interpretation of the results is sometimes complicated. Indeed, for azoles-echinocandins combinations, both synergistic and additive effect, depending on the study endpoint and the mathematical definitions for the drug interaction effect, have been reported [START_REF] Dannaoui | In vitro evaluation of double and triple combinations of antifungal drugs against Aspergillus fumigatus and Aspergillus terreus[END_REF][START_REF] Cuenca-Estrella | Combined activity in vitro of caspofungin, amphotericin B, and azole agents against itraconazole-resistant clinical isolates of Aspergillus fumigatus[END_REF][START_REF] Meletiadis | Assessing in vitro combinations of antifungal drugs against yeasts and filamentous fungi: comparison of different drug interaction models[END_REF][START_REF] Philip | In vitro synergy testing of anidulafungin with itraconazole, voriconazole, and amphotericin B against Aspergillus spp. and Fusarium spp[END_REF][START_REF] Jeans | Combination of voriconazole and anidulafungin for treatment of triazole-resistant aspergillus fumigatus in an in vitro model of invasive pulmonary aspergillosis[END_REF][START_REF] Planche | In vitro combination of anidulafungin and voriconazole against intrinsically azolesusceptible and -resistant Aspergillus spp[END_REF][START_REF] Seyedmousavi | Efficacy and pharmacodynamics of voriconazole combined with anidulafungin in azole-resistant invasive aspergillosis[END_REF][START_REF] Mavridou | The strength of synergistic interaction between posaconazole and caspofungin depends on the underlying azole resistance mechanism of Aspergillus fumigatus[END_REF][START_REF] Raffetin | In vitro combination of isavuconazole with echinocandins against azole-susceptible and -resistant Aspergillus spp[END_REF].. For these reasons, in addition to in vitro studies, we used an in vivo model to assess the combinations. This model was previously used and validated for the evaluation of treatment of aspergillosis [START_REF] Forastiero | In vivo efficacy of voriconazole and posaconazole therapy in a novel invertebrate model of Aspergillus fumigatus infection[END_REF][START_REF] Maurer | Galleria mellonella as a host model to study Aspergillus terreus virulence and amphotericin B resistance[END_REF][START_REF] Jemel | Galleria mellonella for the evaluation of antifungal efficacy against medically important fungi, a narrative review[END_REF][START_REF] Jemel | In vivo efficacy of voriconazole in a Galleria mellonella model of invasive infection due to azole-susceptible or resistant Aspergillus fumigatus isolates[END_REF]. In a first set of experiments, monotherapies were tested at different dosages to assess their efficacy and to determine the optimal dosage for combination studies. VRZ was previously tested in the same model [START_REF] Jemel | In vivo efficacy of voriconazole in a Galleria mellonella model of invasive infection due to azole-susceptible or resistant Aspergillus fumigatus isolates[END_REF], and it was shown that efficacy was correlated to in vitro susceptibility, and that a dosage of 4 µg/larva would be suitable for combination experiments. In the present study, we further evaluated CAS and PSZ monotherapies. For CAS monotherapy an increased survival was only observed for AfS-infected larvae but without dose dependent efficacy which is in line with a previous animal study [START_REF] Lepak | Impact of in vivo triazole and echinocandin combination therapy for invasive pulmonary aspergillosis: enhanced efficacy against Cyp51 mutant isolates[END_REF].

For PSZ monotherapy, a dose-dependent efficacy was observed for AfS and a lower efficacy against the two PSZ-resistant strains. Nevertheless, a certain degree of efficacy was obtained against the PSZ-resistant strains with a paradoxical better efficacy against the strain with a higher MIC. Although in vitro-in vivo correlation has been reported for PSZ [START_REF] Lepak | Impact of in vivo triazole and echinocandin combination therapy for invasive pulmonary aspergillosis: enhanced efficacy against Cyp51 mutant isolates[END_REF][START_REF] Forastiero | In vivo efficacy of voriconazole and posaconazole therapy in a novel invertebrate model of Aspergillus fumigatus infection[END_REF], discrepancies between in vitro results and in vivo efficacy have also been reported previously. For example, Sun et al. [START_REF] Sun | In vitro and in vivo interactions of TOR inhibitor AZD8055 and azoles against pathogenic fungi[END_REF] evaluated the in vitro and in vivo efficacy of azoles against Af and observed that PSZ improved significantly the survival of G. mellonella larvae infected by a PSZ-resistant strain (MIC of 2 µg/mL). Possible explanations for the efficacy of an antifungal against resistant strains could be the use of high dosages or a lower virulence (fitness-cost) of the resistant strains as shown in the study of dos [START_REF] Reis | The Aspergillus fumigatus mismatch repair MSH2 homolog is important for virulence and azole resistance[END_REF] in which some PSZ resistant mutants derived from a wild type strain lost their virulence.

Nevertheless, in our work, the LD 90 was determined for the three strains and no difference in term of virulence was seen between AfS, AfR1 and AfR2 [START_REF] Jemel | In vivo efficacy of voriconazole in a Galleria mellonella model of invasive infection due to azole-susceptible or resistant Aspergillus fumigatus isolates[END_REF].

Overall, in the present study, the combination of an azole with caspofungin showed both indifferent and synergistic interactions depending on the strain susceptibility.

When compared to VRZ monotherapy, the combination of VRZ with CAS had a better efficacy for the VRZ-resistant strain (i.e. AfR2) infected larvae. This is interesting, as combination therapy is recommended in cases of azole-resistance [START_REF] Verweij | International expert opinion on the management of infection caused by azole-resistant Aspergillus fumigatus[END_REF]. In previous studies, both indifferent (MacCallum et al., 2005;[START_REF] Zhang | Efficacy of the combination of voriconazole and caspofungin in experimental pulmonary aspergillosis by different Aspergillus species[END_REF] and synergistic interactions have been reported [START_REF] Kirkpatrick | Efficacy of caspofungin alone and in combination with voriconazole in a Guinea pig model of invasive aspergillosis[END_REF], but it has to be noticed that most of the studies have been performed with susceptible isolates.

In our study, combination of PSZ at 4 µg/larva and CAS at 4 µg/larva improved the rate of survival in larvae infected by AfR1 (PSZ resistant strain) when compared to CAS or PSZ alone.

These observations are supported by a neutropenic murine model of pulmonary invasive aspergillosis in which efficacy was determined using quantitative PCR [START_REF] Lepak | Impact of in vivo triazole and echinocandin combination therapy for invasive pulmonary aspergillosis: enhanced efficacy against Cyp51 mutant isolates[END_REF].

Combination therapy with CAS and PSZ did not enhance efficacy for PSZ-susceptible isolates.

However, the drug combination produced synergistic activity against PSZ-resistant isolates.

Conclusion

Overall, our results showed relatively weak interactions between azoles and caspofungin against Af in vitro. In vivo, a better efficacy of the combination compared to the azole monotherapy was obtained only against the azole-resistant isolates. Antagonism was never observed. S1; Table S2; Figure S1

Supplementary Materials: Table

  , Figure S2, Figure S3 Author Contributions: Conceptualization, E.D.; methodology, S.J., Y.R., A-L.B., and G.J.; formal analysis, S.J. Y.R., A-L.B., and E.D.; data curation, S.J. A-L.B., and E.D.; writing-original draft preparation, S.J. and E.D.; writing-review and editing, S.J., Y.R., A-L.B., J.G., K.K., G.J., F.B., and E.D.; supervision, E.D.; funding acquisition, F.B., E.D., and J.G. All authors have read and agreed to the published version of the manuscript.

Figure legends Figure 1 :

 1 Figure legends Figure 1: Survival curves of groups of G. mellonella larvae inoculated with AfS (left), AfR1

Figure 2 :

 2 Figure 2: Survival curves of groups of G. mellonella larvae inoculated with AfS (left), AfR1

Figure 3 :

 3 Figure 3: Survival curves of groups of G. mellonella larvae inoculated with AfS (left), AfR1

Figure 4 :

 4 Figure 4: Survival curves of groups of G. mellonella larvae inoculated with AfS (left), AfR1

Table 1 :

 1 in vitro susceptibility of the three Af strains

	Antifungal and method	AfS	MIC (µg/mL) against AfR1	AfR2
	Amphotericin B
	EUCAST	1	1	0.5
	Etest® a	0.25	0.5	0.25
	Itraconazole
	EUCAST	0.25	>4	>4
	Etest®	0.5	>32	32
	Voriconazole
	EUCAST	0.25	0.5	2
	Etest®	0.125	0.125	1
	Posaconazole
	EUCAST	0.06	>4	0.5
	Etest®	0.06	>32	0.5
	Caspofungin
	EUCAST	0.25	0.5	0.5
	Etest®	0.032	0.008	0.032
	a GCS was determined at 48h for AfS and AfR1, and at 24h for AfR2

Table 2 :

 2 In vitro interaction between CAS and VRZ by checkerboard

		MIC (µg/mL) of	MIC (µg/mL) of drug in	Lowest FICI for the
	Isolate	drug alone	combination		combination
		CAS	VRZ	CAS	VRZ	CAS/VRZ	Interaction
	AfS	1	0.25	0.0156	0.25	1.0156	I
	AfR1	1	0.5	0.25	0.25	0.75	I
	AfR2	1	2	0.25	1	1.25	I
	MIC: Minimal Inhibitory Concentration; FICI: Fractional Inhibitory Concentration Index; CAS: caspofungin; VRZ:
	voriconazole; I: no interaction.

Table 3 :

 3 In vitro interaction between CAS and PSZ by checkerboard

		MIC (µg/mL) of	MIC (µg/mL) of drug in	Lowest FICI for the
	Isolate	drug alone	combination	combination
		CAS	PSZ	CAS	PSZ	CAS/PSZ	Interaction
	AfS	1	0.125	0.0156	0.0625	0.5156	I
	AfR1	1	8	ND	ND	ND	ND
	AfR2	1	1	0.015625	0.5	0.5156	I
	MIC: Minimal Inhibitory Concentration; FICI: Fractional Inhibitory Concentration Index; CAS: caspofungin; PSZ:
	posaconazole; I: no interaction. ND: not determined (all MICs > maximum).

Table 4 :

 4 In vitro interaction between CAS and azoles evaluated by a Bliss independence-

	based model
	Isolate	ƩSSI	CAS/VRZ combination MSSI (95% CI) Interaction	ƩSSI	CAS/PSZ combination MSSI (CI 95%) Interaction
	AfS	50.3% 3.9% (-2.3;10.0)	I	185.5%	23.2% (-2.6;49.0)	I
	AfR1 747.5% 37.4% (30.1;44.6)	S	1195.3% 36.2% (30.3;42.2)	S
	AfR2	89.5% 6.9% (-4.3;18.1)	I	-12.7%	-4.2% (-23.2;14.7)	I
	ƩSSI: Sum of statistically significant interactions MSSI: Mean of statistically significant interactions; 95% CI: Confidence
	interval at 95% level; CAS: caspofungin; VRZ: voriconazole; PSZ: posaconazole; S: synergy; I: no interaction.

Table S2 :

 S2 In vitro interaction between CAS and PSZ by gradient concentration strips

	Isolate	MIC (µg/mL) of drug alone	MIC (µg/mL) of drug in combination	FICI for the combination
		CAS	PSZ	CAS+PSZ	CAS +PSZ	Interaction
	AfS	0.032	0.094	0.064	2.67	I
	AfR1	0.006	>32	0.006	1.00	I
	AfR2	0.016	0.5	0.016	1.03	I
	MIC: Minimal Inhibitory Concentration; FICI: Fractional Inhibitory Concentration Index; CAS: caspofungin; PSZ:
	posaconazole; I: no interaction.

Funding: This research received funding from MSD (Investigator Initiated Studies Program n° 57288).

Institutional Review Board Statement: Not applicable.

Data Availability Statement:

The data presented in this study are available on request from the corresponding author.

Conflicts of Interest:

During the past 5 years, Eric Dannaoui has received research grants from MSD and Gilead; travel grants from Gilead, MSD, Pfizer, and Astellas, and speaker's fee from Gilead, MSD, and Astellas. Françoise Botterel has received research grants from MSD; travel grants from Gilead, MSD, Pfizer, and speaker's fee from Gilead, MSD, and Pfizer. Jacques Guillot has received research grants from MSD Animal Health and Vetoquinol, and speaker's fee from Boehringer Animal Health, Elanco, Gilead, Cerbavet and Virbac.

Supplementary material

Supplementary Figure legends


Ali Alaeldine
email: ali.alaeldine@eseo.fr

Thomas Ordas

Philippe Richard Perdriau #

Mohamed Ramdani #

Lionel Torres

M 'hamed Drissi

Assessment of the Immunity of Unshielded Multi-Core Integrated Circuits to Near-Field Injection

This paper presents a comparative assessment of the electromagnetic immunity of 4 integrated logic cores to near-field injection. These cores, located on the same die, are identical from a functional point of view, but differ by their design strategies. The injection is performed above each core according to the 6 components of the electromagnetic field, using appropriate probes. These results demonstrate that the die and bondwires of an integrated circuit can be sensitive to both magnetic and electric fields, and that some design rules can improve the immunity of integrated circuits to near-field interference.

I. INTRODUCTION

Within the recent years, many digital and analog integrated circuits (ICs) have become more and more susceptible, due to an increased number of interfaces, higher data rates, decreased node capacitance, and a steady reduction in power supply voltage and, consequently, noise margin. In order to characterize the electromagnetic behavior of these ICs, several measurement methods have been developed by the International Electrotechnical Commission (IEC), in conducted or radiated mode. These methods include near-field [1]; however, this standard is (at the time when this paper is written) limited to emission, although near-field can be used for immunity as well, by injecting power into the pins of an IC through a nearfield probe and observing its behavior according to a given criterion [2] [3]. This method is currently under standardization process by the IEC under the 62132-9 reference. In this paper, this method is used in order to assess the immunity of unshielded ICs (packaged but without any lid) to electric and magnetic near-field. Furthermore, comparisons among several electromagnetic interference (EMI) protection techniques are achieved using a multi-core IC (CESAME) [START_REF] Vrignon | Characterization and modeling of parasitic emission in deep submicron CMOS[END_REF] which had already been used for such evaluations in conducted mode [START_REF] Alaeldine | Efficiency of embedded on-chip EMI protections to continuous harmonic and fast transient pulses with respect to substrate injection[END_REF]. By minimizing the parasitic influence of packaging, a better comprehension of the immunity of the die itself can be achieved. The paper is organized as follows. First of all, the internal structure of the CESAME test chip is introduced in Sect. II. Then, Sect. III presents the near-field measurement set-up used in this study. Finally, Sect. IV provides a summary of all measurement results, along with a discussion on the causes of the observed phenomena.

II. TEST CHIP

A. General structure

The integrated circuit used in this study (CESAME) was designed and fabricated by STMicroelectronics in 0.18 µm CMOS technology (1.8 V supply voltage), and was initially intended for the validation of low-emission design techniques. It consists of six logic cores which are identical from a functional point of view and are located on the same die, but differ only by their protection strategies. All these cores are based on D flip-flops, a clock tree and standard gates, and are intended to reflect the activity of a typical logic core [START_REF] Vrignon | Characterization and modeling of parasitic emission in deep submicron CMOS[END_REF]. Each core includes 240 identical synchronous base cells, and each base cell consists of 400 transistors (5 D-flip-flops, 25 NAND gates and 4 buffers). 4 cores (NORM, RC, NOR and GRID) are built in the global substrate of the IC, while the other ones (ISO and ISV) are built in a local isolated substrate; for the sake of simplicity, only 4 different cores (NORM, GRID, ISO and RC) out of 6 are used. The IC is mounted in a 144-pin TQFP package. Courtesy of STMicroelectronics, packages with removable lids were used, making it possible to inject a radiated perturbation into a given core with accurate positioning and minimum parasitic influence of the package.

B. NORM core

The only EMI protection strategy used in the NORM core consists of two small 1.7 Ω series resistors, one on each power supply rail. These resistors, along with the metal and MOS capacitances of the logic core, build up a RC filter, with a high cutoff frequency (about 200 MHz).

C. ISO core

Another protection strategy is used for the ISO core. This core is embedded in its own local substrate, isolated from the rest of the chip thanks to a triple-well technique (Fig. 1).

D. GRID core

The GRID core, like the NORM core, only uses the series resistors as a protection technique. However, its power supply network is meshed on the lower metal levels (hence the name), thus reducing the total power supply impedance.

E. RC core

In this core, an additional 1-nF integrated decoupling capacitor is included between both supply rails. This distributed on-chip capacitor is made from several poly1/poly2 capacitors, and increases the area of the RC core by 40 % compared with the NORM core. By lowering the cutoff frequency of the RC filter (about 40 MHz), this technique makes it possible the power distribution noise arising from multiple drivers switching simultaneously.

III. NEAR-FIELD INJECTION SET-UP

The measurement set-up chosen for the characterization of the susceptibility of an IC to near-field interference closely mimics the Direct Power Injection (DPI) set-up. It is depicted in Fig. 2. For this experiment, the IC is mounted on a 10x10 cm 4-layer PCB [START_REF] Vrignon | Characterization and modeling of parasitic emission in deep submicron CMOS[END_REF], powered by a 9 V battery through a 1.8 V regulator, and fed with 20 MHz clock and 10 MHz data signals, in order to ensure normal operation. Then, near-field interference is injected through an adequate probe located at 1 mm above each core. 1 mm diameter loops are used for magnetic field injection, while 3 mm long probes are used for electric field injection. The accurate positioning of the probe is ensured thanks to a picture of the GDS2 layout kindly supplied by STMicroelectronics. Fig. 3 illustrates an electric field injection into the unpackaged IC. This probe is fed by a RF generator and a power amplifier (rated at 10 W, but delivering up to 46 dBm depending on frequency) through a directional coupler, allowing the measurement of incident and reflected powers through a dualchannel power meter. Therefore, an inductive or a capacitive coupling (depending on the probe) is established between with the core. It can be noted that the bandwidth of the amplifier unfortunately limits measurements to 1 GHz. The immunity criterion is an ubiquitous one issued from CAN transceiver testing [START_REF]Integrated circuits -EMC evaluation of CAN transceivers[END_REF] and already used in many papers. The operation of the IC is considered defective as soon as the ripple of the output amplitude crosses the 20 % margin or the jitter of the output signal is higher than 10 %.

IV. RESULTS AND DISCUSSION

Measurements were performed on the 4 cores (NORM, ISO, GRID and RC) for each component of the electromagnetic field (E x , E y , E z , H x , H y , H z ), from 10 MHz to 1 GHz in 10 MHz steps. For each frequency, the injected power triggering a malfunction, according to the aforementioned criterion, was recorded and used for subsequent plots. During this experiment, it was observed that, below approximately 200 MHz, interference has an influence on ripple whereas, above this limit, jitter is affected. The first result obtained demonstrated that all cores are immune to a 46 dBm injection (upper limit of the amplifier) in normal magnetic field (H z ) at any frequency up to 1 GHz. Therefore, no figure is supplied for this component. The high immunity of all cores to the H z field comes from the small dimensions of horizontal metal loops on the die; in fact, the voltage disturbance induced in these loops by the probe is very low. The NORM core, which is the most susceptible core among those studied, is less immune to H x than to H y . The explanation may come from the coupling between the magnetic field emitted by the probe and the power supply bondwires: when the loop is parallel to these bondwires, field lines induce a much higher voltage in the power supply than in the case of a perpendicular loop; these disturbances are then reinjected into the die in conducted form. The fact that the RC core is the most immune to magnetic fields may confirm this assumption and explain why this result matches the one observed in direct power injection (DPI) [START_REF] Alaeldine | Efficiency of embedded on-chip EMI protections to continuous harmonic and fast transient pulses with respect to substrate injection[END_REF]. Furthermore, it is likely that the die itself is not susceptible to tangential magnetic fields, since the area of vertical loops (in the power distribution network) is very small. In general, all cores are more susceptible to electric fields than to magnetic fields. In fact, integrated circuits use "body ties", namely substrate to GND or N-well to VDD contacts, designed to minimize voltage gradients in the substrate. These body ties build up large metal zones with a very low impedance towards the power supply, onto which an electric field can couple itself quite easily (and to a greater extent in the case of a vertical electric field). This capacitive coupling may also explain the difference observed with the GRID core; the capacitive coupling with the meshed power supply network causes new resonance and antiresonance frequencies. Both RC and ISO cores bear almost the same immunities to E x and E y ; however, as far as the E z field is concerned, the ISO core is the least susceptible. From this result, an assumption may be made. The vertical electric field is mainly coupled to the body ties, and its reinjection into the ISO core is minimal due to the lower equivalent capacitance of its substrate; conversely, the tangential electric field also targets bondwires and injects the disturbance into the core through the power supply network, hence being filtered by the integrated decoupling capacitance. In order to identify the contribution of the probes themselves to these results, the standing wave ratios (SWR) of these probes were measured with and without the board under test, with no significant differences. Fig. 9 and 10 depict the SWR of the electric probe and the magnetic probe. As can be seen, the SWR decreases from 30 to 18 (E probe) or 20 to 12 (H probe) in the 0.4 -1 GHz range, but rather steadily and with only small variations with respect to its absolute value. Consequently, the steep immunity "valleys" observed in the previous figures are not due to the probes themselves.

V. CONCLUSION

In this paper, the effect of near-field electromagnetic injection on unshielded ICs was studied. In particular, comparisons among different EMI protection strategies on logic cores were assessed. It was demonstrated that integrated circuits are sensitive not only to magnetic field, but also (and to a greater extent) to electric field; moreover, the susceptibility of an integrated circuit to radiated emission does not come only from its bondwires and its package, but also from the die itself. In most cases (except for normal electric field), the integrated decoupling capacitor was proven to be a very valuable technique for susceptibility reduction, as it had been demonstrated in the past for direct power injection (DPI).

In order to evaluate the influence of packaging with better accuracy, comparisons will be performed with commercially packaged integrated circuits (the package acting like an electromagnetic shield). It should then be possible to enhance the existing electrical models of near-field injection with better coupling models.

Fig. 1 .

 1 Fig. 1. Comparison between NORM and ISO core architectures

Fig. 2 .Fig. 3 .

 23 Fig. 2. Near-field injection set-up

Fig. 4 .Fig. 5 .

 45 Fig. 4. Immunity of the 4 cores to the Hx field

Fig. 6 .Fig. 7 .

 67 Fig. 6. Immunity of the 4 cores to the Ex field

Fig. 8 .

 8 Fig. 8. Immunity of the 4 cores to the E z field

Fig. 9 .

 9 Fig. 9. SWR of the electric probe

  Fig. 10. SWR of the magnetic probe