Jérémie Bettoni

Matthieu Olivetto

Jebrane Bouaoud

M.D. d , Stéphanie Dakpé M.D. Ph.D Jérôme Duisit

Stéphanie Dakpé

Bilateral bifid condyles: A rare etiology of temporomandibular joint disorders

Keywords: temporomandibular joint, bifid mandibular condyle, temporomandibular dysfunction, management

Text 1. Introduction

Described for the first time by Hrdlicka [START_REF] Hrdlicka | Lower Jaw: double condyles[END_REF] as a condylar split or groove of variable depth, bifid mandibular condyle (BMC) is an extremely rare temporomandibular joint (TMJ) disorder with a prevalence of 0.018% to 1.82% [START_REF] Menezes | The prevalence of bifid mandibular condyle detected in a Brazilian population[END_REF][START_REF] Sahman | Detection of bifid mandibular condyle using computed tomography[END_REF]. BMC can be asymptomatic or present non-specific clinical signs and symptoms such as pain, swelling, noise, hypomobile joint blockage, deflection, joint luxation, or even ankylosis [START_REF] Borras-Ferreres | Bifid mandibular condyles: A systemic review[END_REF]. The diagnosis of BMC is most often made after an incidental finding on a panoramic radiograph during a routine exam and confirmed by computed tomography (CT), considered as the gold standard for confirmation of its diagnosis [START_REF] Cowan | Bifid mandibular condyle[END_REF][START_REF] De Sales | Bifid mandibular condyle: Case report and etiological consifderations[END_REF][START_REF] Balaji | Bifid mandibular condyle: A study of the clinical features, patterns and morphological variations using CT scans[END_REF].

Case Report

The authors report the case of a 20-year-old female patient, with no relevant past medical or surgical history, coming to the maxillofacial department complaining of chronic pain from both TMJ, with progressive worsening in the previous months without any objective triggering factor. The clinical examination revealed pain upon TMJ palpation, predominantly on the right side, with associated audible clicking as well as a limited mouth opening measuring 21 mm, a right lateral deviation, and inability to perform mandibular propulsion (Figure 1). To rule out an organic etiology, a panoramic radiograph (Figure 2a) was performed and showed an atypical deformity of the left mandibular condyle requiring additional investigation. A dynamic X-ray CT scan with open and closed mouth positions confirmed the diagnosis of bilateral BMC with mediolateral orientation of the condyle head and a predominant TMJ abnormality on the right side explaining the clinical features (Figure 2b and2c).

Considering the progressive worsening of symptoms (pain, limited mouth opening on the right side, and ankylosis), the authors decided to perform a bilateral arthrotomy with the resection of lateral articular condyle surfaces with Piezotome surgery through a preauricular bilateral approach (Figure 3). The TMJ exploration revealed a healthy disc, with no degenerative lesions or visible perforations. Pathology findings of the resected lateral joint surfaces were in favor of the excision of healthy condylar heads without suspicion of malignancy, with bone spans and cartilage surfaces demonstrating completely normal architecture and thickness.

At one month post surgery, the clinical examination showed an improvement in mouth opening (measuring 3 cm), restoration of mandibular propulsion, and correction of deviation during mouth opening on the right side. After 12 months post operative and 9 months of active rehabilitation, the patient no longer reported pain, clicking, or limitation of the mouth opening (measuring more than 5 cm) (Figure 4).

Discussion

BMC is a rare etiology of TMJ disorders [START_REF] Menezes | The prevalence of bifid mandibular condyle detected in a Brazilian population[END_REF][START_REF] Sahman | Detection of bifid mandibular condyle using computed tomography[END_REF], and awareness and knowledge about this condition is essential for better understanding of its pathophysiological process and its potential progression. This will allow clinicians to establish an appropriate therapeutic management system. The increasing number of papers on BMC bears witness to its importance and confirms the difficulty in determining its etiopathogenesis and impact.

Although BMC can be unilateral or bilateral (ratio 2:1), in 1990 Szenpetery et al. [START_REF] Szentpetery | The problem of bifid mandibular condyle[END_REF] suggested that the anteroposterior position of the condyles most likely results from early childhood fractures, while the mediolateral condyle orientation was associated with a nontraumatic etiology, probably due to persistent developmental septa. However, the description of post-traumatic mediolateral BMC clearly questions this theory. In a recent meta-analysis, Guven [START_REF] Güven | A study on etiopathogenesis and clinical features of multi-headed (bifid and trifid) mandibular condyles and review of the literature[END_REF] identified two types of BMC. The author proposed differentiating between a type I non-traumatic, non-symptomatic mediolateral BMC from a type II with a history of mandibular trauma to the anteroposterior or mediolateral BMC. This study subdivided type II into 2 groups: a) with a Y-shaped condyle secondary to an intracapsular or vertical condyle trauma; and b) with two separate and anteroposteriorly located condyles, where the formation of the anteroposterior condyle might be due to insufficient remodeling of the ramus/condyle of the displaced or dislocated subcondylar fracture resulting from force exerted by the lateral pterygoid muscle. However, for Dennison et al. [START_REF] Dennison | The false and the true bifid condyles[END_REF], rather than the orientation of the articular surfaces of the BMC, clinical aspects should be favored. They preferred to refer to condylar notching , for asymptomatic BMC with a history of incidental discovery, and true BMC with a clinical impact: pain, clicking, limited freedom of mandibular movement, in some cases progressing to true mandibular ankylosis. This classification is more relevant, as it makes it possible to guide the therapeutic strategy which favors foregoing therapeutic treatment and instead recommends regular monitoring in the case of asymptomatic BMC. Stepwise management is recommended in the case of true BMC guided by the clinical symptoms. Thus, symptomatic treatment with non-steroidal antiinflammatory drugs, analgesics, physiotherapy, or an occlusal splint is recommended as a conservative approach [START_REF] Antoniades | Bilateral bifid mandibular condyle. Oral Surg Oral Med Oral[END_REF][START_REF] Almasan | Nontraumatic bilateral bifid condyle and intermittent joint lock: a case report and literature review[END_REF] in case of pain, creaking, or swelling. For hypomobility, severe pain, or ankylosis, surgical treatment [START_REF] Gulati | Bifid mandibular condyle with temporomandibular joint ankylosis: report of two cases and review of literature[END_REF][START_REF] To | Supero-lateral dislocation of sagittally split bifid mandibular condyle[END_REF][START_REF] Jha | Posttraumatic bifid and trifid mandibular condyle with bilateral temporomandibular joint ankylosis[END_REF] (arthroscopy, arthrotomy, or condylectomy with reconstruction with TMJ prosthesis or costal graft) is described, with the aim of restoring function. In this case, the severe symptomatology and mandibular hypomobility make it difficult to envisage functional treatment. Thus, in the current study, the authors opted for bilateral arthrotomy of the lateral articular surfaces.

The choice of the preauricular pretragal approach may be opposed to other condylar approaches (submandibular, retroauricular, or endobuccal). Nevertheless, it offers the advantage of complete exposure of the TMJ with minimal esthetic sequelae and no risk of secondary ear canal stenosis. Its use requires careful dissection in the auricular perichondral plane until contact with the zygomatic arch, then moving towards the TMJ. This dissection under the plane of the facial nerve limits the risk of nerve damage. It is nevertheless important to control the force exerted on the parotid tissue to avoid any stretching of the nerve branches.

Conclusion

Bifid condyle is a relatively rare condition whose prevalence is increasing, and it requires clinicians to rule out a probable misdiagnosis and understand its real pathophysiology. In these cases, it seems important to propose a precise examination and to complete the assessment with an X-ray CT scan. Regarding its management: -In the event of incidental discovery, regular monitoring is required to ensure appropriate early management.

-In cases with a clinical impact, bilateral arthrotomy using the preauricular approach is a functional and esthetic surgical alternative provided that a rigorous active physiotherapy plan is followed.

Captions to illustrations

Figure 1 :

 1 Figure 1: Clinical view of mandibular hypomobility and deviation at the mouth opening on

Figure 2 :

 2 Figure 2: (a) Panoramic radiograph and (b-c) 3D volume rendering reconstruction of the X-

Figure 3 :

 3 Figure 3: 3D volume rendering reconstruction of the postoperative X-ray CT scan of the

Figure 4 :

 4 Figure 4: Clinical view at 12 months post surgery and after 9 months of active physiotherapy

Figure 1 CFigure 2 C

 12 Figure 1

Figure 3 C

 3 Figure 3

Figure 4 C

 4 Figure 4

Conflict of interest statement:

The authors declare that they have no competing interest.

Financial disclosure:

None

Human rights statements and informed consent:

The patient's informed consent has been obtained for the use of his photos and personal data.


M.D Jebrane Bouaoud
email: jebrane.bouaoud@gmail.com

Matthieu Olivetto

Jeremie Bettoni

Bernard Devauchelle

Muscle entrapment in Orbito-zygomatico-maxillary complex fracture

Keywords: Orbito-zygomatic fracture, Ophthalmologic injury, Muscle entrapment, Clean_Revised_Manuscript Orbito-zygomatic fracture, Ophthalmologic injury, Muscle entrapment, Track_changes_Revised_Manuscript

Case report

A 48-year-old man without relevant personal medical history, was addressed to the emergency department for medical care following a maxillofacial trauma after receiving a stone on the left cheekbone. The general examination revealed an alcohol-related trauma without loss of consciousness. The patient was alert, Glasgow 14 and presented a spontaneously resolved epistaxis.

At the maxillofacial examination, we have found a middle face trauma with a deformation of the left hemiface due to bones displacement, a left peri orbital hematoma, photophobia, a very painful limitation of all ocular movement of the left eye (Figure 1, supplementary video) and diplopia. There was no loss of vision. The mouth opening was painfully limited to 1.5 centimeters. The rest of the maxillofacial examination was unremarkable. The patient was not comforted by opioid analgesics. A computerized tomography (CT scan) was realized (Figure 2).

What is your diagnosis?

Answer

Left orbito-zygomatico-maxillary complex (OZMC) fracture with lateral wall displacement and entrapment of the lateral and inferior rectus muscles, without intracranial lesion but with an undisplaced and asymptomatic fracture of the left mandibular coronoid process.

Immediate surgery under general anesthesia was performed. An ophthalmic exam was realized at the beginning of the procedure. No corneal or intra-orbital lesions were found. Then, we have realized a forced duction test which has confirmed the limitation of the extra ocular motility for elevation an adduction of the left globe. All the fracture sites were controlled by three surgical approaches (oral, sub ciliary and sub brow). After liberation of oculomotor muscles, we have realized an open reduction and osteosynthesis by mini-plaques (Modus® 1.5) of the different fracture sites (Figure 3). At the end of the surgery, the forced duction test showed no entrapment. The postoperative period was uneventful, and an ophthalmologic control confirmed the absence of trouble.

Discussion

Lateral orbital wall fractures are rarely isolated but most commonly seen with orbito-zygomaticomaxillary complex (OZMC) fractures. These fractures account for around 27% of all facial fractures 1 .

Alcohol-related assault for males are the most common presentation of this type of injury.

Restriction of the extraocular muscles is among the most frequent ophthalmic injuries associated with fractures of the OZMC, reaching up to 15% of cases 2 . There is a real benefit of early preoperative detection of ophthalmological injuries to manage it as soon as possible. The delay between the trauma and the treatment is a prognostic factor for ophthalmologic outcomes. Furthermore, it is important from a medicolegal position 2 . Thus, entrapped muscle with limitation of motility is an indication of immediate surgical management 3 . The maxillofacial surgeons should have a thorough knowledge of the various ophthalmologic injuries that could occur secondary to facial trauma 4 .

Computed tomography (CT) is the gold standard for the diagnosis, the evaluation of the complexity of the OZMC fracture and allow to research complications such as globe, optic nerve and muscle injuries. In addition, the CT provides information on neurologic status to rule out any neurological cause of confusion in an alcoholic patient (skull fracture, extra-sub dural hematoma or intracranial bleeding). The evaluation of the sphenozygomatic suture provides a good estimation of the degree of displacement of the lateral wall. Significant displaced bones in OZMC fractures should be reduced and fixed to preserve the anatomical shape of the orbit and to preserve their function of globe protectors 5 .

In all cases, the CT should not delay the treatment. The diagnosis of muscle entrapment is first of all clinic with typically a painful limitation of the extraocular muscles in a context of facial trauma. The clinical examination should be systematized to ensure to not omit brain or eyeball lesions. The alcohol and the post traumatic confusion should not be underestimated.

Case report

A 48-year-old man without relevant personal medical history, was addressed to the emergency department for medical care following a maxillofacial trauma after receiving a stone on the left cheekbone. The general examination revealed an alcohol-related trauma without loss of consciousness. The patient was alert, Glasgow 14 and presented a spontaneously resolved epistaxis.

At the maxillofacial examination, we have found a middle face trauma with a deformation of the left hemiface due to bones displacement, a left peri orbital hematoma, photophobia, a very painful limitation of all ocular movementtricity of the left eye (Figure 1, supplementary video) and diplopia.

There was no loss of vision. The mouth opening was painfully limited to 1.5 centimeters. The rest of the maxillofacial examination was unremarkable. The patient was not comforted by opioid analgesics. A computerized tomography (CT scan) was realized (Figure 2).

What is your diagnosis?

Answer

Left oOrbito-zygomatico-maxillary complex (OZMC) fracture with lateral wall displacement and entrapment of the lateral and inferior rectus muscles, without intracranial lesion but with an undisplaced and asymptomatic fracture of the left mandibular coronoid process.

Immediate surgery under general anesthesia was performed. An ophthalmic exam was realized at the beginning of the procedure. No corneal or intra-orbital lesions were found. Then, we have realized a forced duction test which has confirmed the limitation of the extra ocular motility for elevation an adduction of the left globe. The allAll the fracture sites were controlled by three surgical approaches (oral, sub ciliary and sub brow). After liberation of oculomotor muscles, we have realized an open reduction and osteosynthesis by mini-plaques (Modus® 1.5) of the different fracture sites (Figure 3). At the end of the surgery, the forced duction test showed no entrapment. The postoperative period was uneventful, and an ophthalmologic control confirmed the absence of trouble.

Discussion

Immediate surgery under general anesthesia was performed. An ophthalmic exam was realized at the beginning of the procedure. No corneal or intra-orbital lesions were found. Then, we have realized a forced duction test which has confirmed the limitation of the extra ocular motility for elevation an adduction of the left globe. The all fracture sites were controlled by three surgical approaches (oral, sub ciliary and sub brow). After liberation of oculomotor muscles, we have realized an open reduction and osteosynthesis by mini-plaques (Modus® 1.5) of the different fracture sites (Figure 3). At the end of the surgery, the forced duction test showed no entrapment. The postoperative period was uneventful, and an ophthalmologic control confirmed the absence of trouble.

Lateral orbital wall fractures are rarely isolated but most commonly seen with orbito-zygomaticomaxillary complex (OZMC) fractures. These fractures account for around 27% of all facial fractures 1 .

Alcohol-related assault for males are the most common presentation of this type of injury.

Restriction of the extraocular muscles is among the most frequent ophthalmic injuries associated with fractures of the OZMC, reaching up to 15% of cases 2 . There is a real benefit of early preoperative detection of ophthalmological injuries to manage it as soon as possible. The delay between the trauma and the treatment is a prognostic factor for ophthalmologic outcomes. Furthermore, it is important from a medicolegal position 2 . Thus, entrapped muscle with limitation of motility is an indication of immediate surgical management 3 . The maxillofacial surgeons should have a thorough knowledge of the various ophthalmologic injuries that could occur secondary to facial trauma 4 .

Computed tomography (CT) is the gold standard for the diagnosis, the evaluation of the complexity of the OZMC fracture and allow to research complications such as globe, optic nerve and muscle injuries. In addition, the CT provides information on neurologic status to rule out any neurological cause of confusion in an alcoholic patient (skull fracture, extra-sub dural hematoma or intracranial bleeding). The evaluation of the sphenozygomatic suture provides a good estimation of the degree of displacement of the lateral wall. Significant displaced bones in OZMC fractures should be reduced and fixed to preserve the anatomical shape of the orbit and to preserve their function of globe protectors 5 .

In all cases, the CT should not delay the treatment. The diagnosis of muscle entrapment is first of all clinic with typically a painful limitation of the extraocular muscles in a context of facial trauma. The clinical examination should be systematized to ensure to not omit brain or eyeball lesions. The alcohol and the post traumatic confusion should not be underestimated.

Figure 1 :

 1 Figure 1: Patient pre-operative ophthalmologic examination. Here, we can see a painful limitation

Figure 2 :

 2 Figure 2: Pre-operative X-Ray computed tomography, 3D reconstruction and 2D axial and coronal

Figure 3 :

 3 Figure 3: Post-operative X-Ray computed tomography, 3D reconstruction and 2D axial and coronal

Figure 1 :

 1 Figure 1: Patient pre-operative ophthalmologic examination. Here, we can see a painful limitation

Figure 2 :

 2 Figure 2: Pre-operative X-Ray computed tomography, 3D reconstruction and 2D axial and coronal

Figure 3 :

 3 Figure 3: Post-operative X-Ray computed tomography, 3D reconstruction and 2D axial and coronal

Figure 1

 1 Figure 1 Click here to download high resolution image

Figure 2

 2 Figure 2 Click here to download high resolution image

Figure 3

 3 Figure 3 Click here to download high resolution image


Sylvain Le Tonquesse

Éric Alleno

Valérie Demange

Carmelo Prestipino

Olivier Rouleau

Mathieu Pasturel
email: mathieu.pasturel@univ-rennes1.fr

Reaction mechanism and thermoelectric properties of In 0.22 Co 4 Sb 12 prepared by magnesiothermy

Keywords: Skutterudites, Magnesioreduction synthesis, Reaction mechanism, Thermoelectric properties

The magnesioreduction synthesis of In 0.22 Co 4 Sb 12 with high In-rattler concentration from Sb 2 O 4 and In-doped Co 3 O 4 precursors is reported. This process directly yields a submicronic powder in a single step of 96 h at 810 K. The reaction mechanism has been investigated by stopping the reaction every 12 h and quantifying the existing phases by X-ray diffraction and Rietveld refinements. The precursors are first reduced in CoO and Sb 2 O 3 lower oxides, then form CoSb 2 O 6 and CoSb 2 O 4 intermediates which are finally reduced in In x Co 4 Sb 12 . A powder with 350 nm average size and mostly composed of In-filled skutterudite phase with composition close to In 0.17 Co 4 Sb 12 is obtained. Upon spark plasma sintering, small residual amount of InSb reacts with the skutterudite matrix to form a single-phase densified pellet with composition close to In 0.22 Co 4 Sb 12 .

The resulting densified material with 1.8 µm average grain size shows a figureof-merit ZT max of 0.95 at 750 K.

Introduction

Thermoelectric (TE) materials enable the direct conversion of heat into electricity through the Seebeck effect. The conversion efficiency is directly related to the figure-of-merit ZT defined as:

ZT = α 2 ρ (κ L + κ e ) T (1)
with α the Seebeck coefficient, ρ the electrical resistivity, κ L and κ e the lattice and electronic contributions to the total thermal conductivity κ and T the temperature. CoSb 3 -based skutterudites are well-known TE materials with a band gap of about 0.2 eV [START_REF] Rogl | Skutterudites, a most promising group of thermoelectric materials[END_REF]. They are considered as promising candidates for midtemperature (600 -800 K) TE applications because of their excellent electronic properties, good mechanical properties and relatively abundant constituting elements. Large power factors, PF = α 2 /ρ, reaching 4 -5 mW m -1 K -2 can be obtained for compositions with optimized charge carrier concentration. In order to achieve high ZT, the intrinsic high thermal conductivity (≈ 9 W m -1 K -1 at 298 K for CoSb 3 [START_REF] Leszczynski | Electronic band structure, magnetic, transport and thermodynamic properties of In-filled skutterudites In x Co 4 Sb 12[END_REF]) must be reduced as much as possible without simultaneously degrading the electronic properties. This can be effectively done by partially filling the empty isocahedral voids of the structure with rare-earth, alkaline metal or III -IV group elements [START_REF] Rogl | Skutterudites, a most promising group of thermoelectric materials[END_REF]. The additional mass contrast induced by the partial occupancy as well as the 'rattling' behavior of the filling atoms in the oversized voids effectively scatter acoustic heat-carrying phonons.

For example, lattice thermal conductivities as low as 1 -2 and ≈ 0.5 W m -1 K -1

were reported for single Ba-, Ca-and Yb-filled [START_REF] Li | High pressure synthesized Ca-filled CoSb 3 skutterudites with enhanced thermoelectric properties[END_REF][START_REF] Kang | High pressure synthesis and thermoelectric properties of Ba-filled CoSb 3 skutterudites[END_REF][START_REF] Nolas | High figure of merit in partially filled ytterbium skutterudite materials[END_REF] and nanostructured multi-filled skutterudites [START_REF] Rogl | In-doped multifilled n-type skutterudites with ZT = 1.8[END_REF], respectively.

Indium is among the most studied filler atom for n-type skutterudites because (i) it acts as an electron donor enabling large increase of PF and (ii) it efficiently reduces κ L due to its heavy atomic mass. While some controversy exists about its true solubility limit (0.16 < x < 0.27) [START_REF] Tang | Phase diagram of In-Co-Sb system and thermoelectric properties of In-containing skutterudites[END_REF][START_REF] Grytsiv | In y Co 4 Sb 12 skutterudite: phase equilibria and crystal structure[END_REF][START_REF] Visnow | On the true indium content of In-filled skutterudites[END_REF], all authors agree that the best performances are obtained by maximizing the In concentration [START_REF] Leszczynski | Electronic band structure, magnetic, transport and thermodynamic properties of In-filled skutterudites In x Co 4 Sb 12[END_REF][START_REF] Mallik | Thermoelectric properties of In z Co 4 Sb 12 skutterudites[END_REF]. ZT max of about 1.1 are usually reported for materials with composition close to In 0.25 Co 4 Sb 12 and synthesized by conventional fusion/solidification processes [START_REF] Benyahia | High thermoelectric figure of merit in mesostructured In 0.25 Co 4 Sb 12 n-type skutterudite[END_REF][START_REF] Leszczynski | Electronic band structure, magnetic, transport and thermodynamic properties of In-filled skutterudites In x Co 4 Sb 12[END_REF]. However, due to the peritectic formation of CoSb 3 , long annealing (> 100 h) at high temperature (1100 K) are required to form a single phase product. In addition, the ingots are composed of microns sized grains which need to be reduced by milling in order to reduce κ L . κ reduction were attempted using such conventional approach by in situ formation of InSb nanoprecipitates [START_REF] Eilertsen | Rattler-seeded InSb nanoinclusions from metastable indium-filled In 0.1 Co 4 Sb 12 skutterudites for highperformance thermoelectrics[END_REF][START_REF] Li | Thermoelectric properties of Indium-added skutterudites In x Co 4 Sb 12[END_REF][START_REF] Li | Grain boundary engineering with nano-scale InSb producing high performance In x Ce y Co 4 Sb 12+z skutterudite thermoelectrics[END_REF] or by designing mesostructured materials with oxide nanoinclusions [START_REF] Benyahia | High thermoelectric figure of merit in mesostructured In 0.25 Co 4 Sb 12 n-type skutterudite[END_REF][START_REF] Chubilleau | Thermoelectric properties of In 0.2 Co 4 Sb 12 skutterudites with embedded PbTe or ZnO nanoparticles[END_REF]. These additional annealing and milling steps, to be performed under unreactive atmosphere, cost time and energy cost and could become an obstacle to the large scale production of this material.

Alternative processes have been developed to overcome the problematic synthesis of this material. For example, hydrothermal [START_REF] Du | Investigation on Indium-Filled Skutterudite Materials Prepared by Combining Hydrothermal Synthesis and Hot Pressing[END_REF][START_REF] Gharleghi | Enhanced ZT of In x Co 4 Sb 12 -InSb nanocomposites fabricated by hydrothermal synthesis combined with solid-vapor reaction: a signature of phonon-glass and electron-crystal materials[END_REF] or melt-spinning/reactive spark plasma sintering approaches [START_REF] Lee | Simple and efficient synthesis of nanograin structured single phase filled skutterudite for high thermoelectric performance[END_REF] have been adapted to the synthesis of nanostructured In x Co 4 Sb 12 with high concentration of interfaces between the grains and lattice thermal conductivity as low as 1.5 W m -1 K -1 . Most importantly, these alternative routes do not require any post-reaction annealing which considerably speed up the synthesis. However, nanostructured materials often suffer from largely higher electrical resistivity usually attributed to the lower density of the sintered materials [START_REF] Toprak | The impact of nanostructuring on the thermal conductivity of thermoelectric CoSb 3[END_REF][START_REF] Du | Investigation on Indium-Filled Skutterudite Materials Prepared by Combining Hydrothermal Synthesis and Hot Pressing[END_REF] or to the enhanced charge carriers scattering at the numerous interfaces/defects, considerably reducing the beneficial effect of the microstructure on ZT.

In a previous article [START_REF] Le Tonquesse | Innovative one-step synthesis of mesostructured CoSb 3 -based skutterudites by magnesioreduction[END_REF], we reported a new magnesioreduction synthesis for In x Co 4 Sb 12 with x = 0.13 according to the reaction:

2 (In)Co 3 O 4 + 9Sb 2 O 4 + 44Mg 810K ---→ 96h 6(In)CoSb 3 + 44MgO (2)
This process, also applied to the production of TE-silicides [START_REF] Le Tonquesse | Mesostructure -thermoelectric properties relationships in V x Mn 1-x Si 1.74 (x = 0, 0.04) higher manganese silicides prepared by magnesiothermy[END_REF], possesses many advantages such as the direct synthesis of high purity skutterudite powders with submicronic average grain sizes without additional long annealing or milling step, high yield, relatively low reaction temperature and the use of cheap and air-stable oxide precursors. Moreover, magnesiothermy is already used to industrially produce metals via the Kroll [START_REF] Kroll | Verformbares Titan und Zirkon[END_REF] or Ames [START_REF] Spedding | Production of Uranium[END_REF] processes and offer thus interesting scalability perspectives. In the present article, this magnesioreduction process is adapted to the synthesis of saturated In 0.22 Co 4 Sb 12 . The discussion will mainly focus on the comprehension of the reaction mechanism which was investigated by X-ray diffraction (XRD). Finally, the thermoelectric properties were measured and compared to those of reference samples from the literature.

Experimental procedures

The detailed procedure for the magnesioreduction synthesis of In-filled skutterudites according to reaction (2) is described in a previous work [START_REF] Le Tonquesse | Innovative one-step synthesis of mesostructured CoSb 3 -based skutterudites by magnesioreduction[END_REF] (a = 8.076 Å [START_REF] Antolini | Synthesis and Thermal Stability of LiCoO 2[END_REF]) suggesting the insertion of In in the structure [START_REF] Ma | Indium-doped Co 3 O 4 nanorods for catalytic oxidation of CO and C 3 H 6 towards diesel exhaust[END_REF]. The pres- the precursors in lower oxides by Mg according to the reactions:

Results and discussion

Reaction mechanism and skutterudite characterizations

123 Co 3 O 4 + Mg 810K ---→ 12h 3 CoO + MgO ∆G r (810 K) = -512.3 kJ mol -1 [29] (3) Sb 2 O 4 + Mg 810K ---→ 12h Sb 2 O 3 + MgO ∆G r (810 K) = -396.4 kJ mol -1 [29] (4)
These reductions are thermodynamically possible at 810 K as indicated by the large negative Gibbs free energy of reaction. In addition, CoSb

The slow diffusion of In in CoSb 3 was already stressed out by Grytsiv et al. [START_REF] Grytsiv | In y Co 4 Sb 12 skutterudite: phase equilibria and crystal structure[END_REF]. After 96 h, the reaction media is mostly composed of In x Co 4 Sb 12 with a = 9.04872(4) Å which corresponds to x = 0.17 -0.18 [START_REF] Tang | Phase diagram of In-Co-Sb system and thermoelectric properties of In-containing skutterudites[END_REF][START_REF] Grytsiv | In y Co 4 Sb 12 skutterudite: phase equilibria and crystal structure[END_REF] It is in agreement with the solubility limit usually reported for In-filled CoSb 3 prepared by conventional melting/annealing [START_REF] Grytsiv | In y Co 4 Sb 12 skutterudite: phase equilibria and crystal structure[END_REF] or solid state diffusion [START_REF] He | Thermoelectric properties of indium-filled skutterudites[END_REF] syntheses. The SEM secondary electron image of the broken cross-section of a pellet is shown in fig. 4a. The microstructure is typical for magnesioreduced skutterudite with well-faceted grains and size distribution characterized by an average value of 1.8 µm. The present grain size is significantly larger than our previous work [START_REF] Le Tonquesse | Innovative one-step synthesis of mesostructured CoSb 3 -based skutterudites by magnesioreduction[END_REF]. This is mostly attributed to the 90 K higher sintering temperature found necessary to complete the reaction. Also the higher content of liquid InSb might have favored grain growth by accelerating matter transport during the sintering [START_REF] Kang | Sintering: densification, grain growth and microstructure[END_REF]. reported by Benyahia et al. [START_REF] Benyahia | High thermoelectric figure of merit in mesostructured In 0.25 Co 4 Sb 12 n-type skutterudite[END_REF] and Leszczynski et al. [START_REF] Leszczynski | Electronic band structure, magnetic, transport and thermodynamic properties of In-filled skutterudites In x Co 4 Sb 12[END_REF] were obtained for materials synthesized by melting/annealing/sintering method. Although the Seebeck coefficient of our sample compares well to literature data, the resistivity is more elevated, especially near room temperature. As a direct consequence, the PF max is 7 to 20 % lower at 750 K. Despite its apparent purity and density, small cracks appeared on our sample upon thermal cycles that could explain the higher measured resistivity. Such deterioration is attributed to the melting around 800 K [START_REF] Grytsiv | In y Co 4 Sb 12 skutterudite: phase equilibria and crystal structure[END_REF] during the measurement cycle of small residual amount of InSb usually observed at the grain boundaries of saturated In-filled skutterudite [START_REF] Eilertsen | Rattler-seeded InSb nanoinclusions from metastable indium-filled In 0.1 Co 4 Sb 12 skutterudites for highperformance thermoelectrics[END_REF][START_REF] Eilertsen | From occupied voids to nanoprecipitates: synthesis of skutterudite nanocomposites in situ[END_REF]. However, the In content in the structure remains constant as indicated by the similar lattice parameter, a = 9.0520(3) Å, determined after the thermoelectric characterization.

Thermoelectric properties

The room temperature thermal conductivity of MR sample is about 3.2 W m -1 K -1

and reaches its minimum value of 2.6 W m -1 K -1 in the 450 -650 K range. The simultaneous decrease of α and upturn of κ at about 650 K is attributed to the bipolar effect i.e. to the contribution of two types of charge carriers to the material transport properties. The lattice thermal conductivity was determined by subtracting κ e to κ and κ e was calculated using the the Wiedemann-Franz law κ e = L T/ρ with L taken from [START_REF] Leszczynski | Electronic band structure, magnetic, transport and thermodynamic properties of In-filled skutterudites In x Co 4 Sb 12[END_REF] as 1. Further comparison of the MR-materials performance with literature data is not a straightforward task as long as the TE properties of In-filled skutterudites strongly depends on the precise In-content inserted in the cages up to its solubility limit [START_REF] Visnow | On the true indium content of In-filled skutterudites[END_REF][START_REF] He | Thermoelectric properties of indium-filled skutterudites[END_REF][START_REF] Li | Thermoelectric properties of Indium-added skutterudites In x Co 4 Sb 12[END_REF][START_REF] Jung | Thermoelectric and transport properties of In-filled CoSb 3 skutterudites[END_REF]. In the present case, we confirm that increasing the In-content from 0.13 [START_REF] Le Tonquesse | Innovative one-step synthesis of mesostructured CoSb 3 -based skutterudites by magnesioreduction[END_REF] [START_REF] Leszczynski | Electronic band structure, magnetic, transport and thermodynamic properties of In-filled skutterudites In x Co 4 Sb 12[END_REF][START_REF] Lee | Simple and efficient synthesis of nanograin structured single phase filled skutterudite for high thermoelectric performance[END_REF] or most recently by scanning laser melting for higher In-content [START_REF] Chen | Scanning laser melting for rapid and massive fabrication of filled skutterudites with high thermoelectric performance[END_REF]. Nevertheless, our ZT value is most of the time higher than those reported after solid state diffusion [START_REF] Peng | Synthesis and thermoelectric properties of In 0.2+x Co 4 Sb 12+x composite[END_REF][START_REF] Biswas | Thermal cycling effects on the thermoelectric properties of n-type In,Cebased skutterudite compounds[END_REF][START_REF] Sesselmann | Neutron diffraction and thermoelectric properties of indium filled In x Co 4 Sb 12 (x = 0.05, 0.2) and indium cerium[END_REF] or HPHT technique [START_REF] Deng | Effects of indium-filling and synthesis pressure on the thermoelectric properties of CoSb 3[END_REF]. Improving the SPS step to limit the grain growth, stabilizing In 0.22 Co 4 Sb 12 /InSb nanocomposites or trying to form multifilled skutterudites by magnesiothermy are the perspectives of this work.

Conclusions

The investigation of the reaction mechanism for the magnesioreduction syn-

Figure 1 :

 1 Figure 1: Pictures of (a) the reaction precursors, (b) reaction products after 96 h at 810 K and (c) crushed as-synthesized In x Co 4 Sb 12 pellets. (d) Secondary electron SEM image of the as-synthesized powder with histogram showing the grain size distribution.

In x Co 4

 4 Sb 12 (targeted x = 0.25) has been synthesized, along with some InSb and Sb impurities, from Sb 2 O 4 and In-doped Co 3 O 4 after 96 h heat treatment at 810 K in presence of Mg. To elucidate the reaction mechanism, the synthesis was repeated several times and stopped every twelve hours. Each time, the phases in the samples were quantified by the Rietveld method and their relative concentrations are represented in fig. 2. Figure 3 shows some selected XRD patterns while all refined patterns and parameters can be found in supplementary information (fig. SI 2, tables SI. 1 -9). Up to 6 different phases have been identified in some patterns indicating a complex reaction mechanism. The full reduction of the precursors is realized in three steps: (i) the partial reduction of the precursors in lower oxides (0 h -24 h), (ii) the formation of CoSb 2 O 6 and CoSb 2 O 4 intermediates (0 h -48 h) and (iii) the complete reduction of the intermediates and the formation of In x Co 4 Sb 12 (38 h -96 h).

Figure 2 :

 2 Figure 2: Evolution of the sample composition during the magnesioreduction synthesis of In x Co 4 Sb 12 , determined by the Rietveld method, as a function of the reaction time.In the present case and according to[START_REF] Toraya | Estimation of statistical uncertainties in quantitative phase analysis using the Rietveld method and the whole-powder-pattern decomposition method[END_REF], the relative standard deviation on the concentrations are considered to be well below 5 %.

Figure 3 :

 3 Figure 3: XRD patterns of the In x Co 4 Sb 12 magnesioreduction synthesis after 0 h, 12 h, 36 h, 96 h reaction time and after spark plasma sintering. The symbols indicate the most intense reflections of each constituting phases. The images shows the evolution of the product color with the reaction time.

  and of a small amount of InSb (≈ 4 mol.%) and Sb (≈ 2 mol.%). No traces of MgO or Mg containing compound are visible at any time on the diffraction patterns. For this reason, we hypothesize that the reduction reactions occur via the slow oxidation of the Mg chunks by the equilibrium O 2 vapor pressure of the oxides. This is consistent with the general aspect of the Mg chunks between 24 h to 48 h which are clearly oxidized at the surface (powdery and white) but remain metallic and relatively shinny in the core.The as-synthesized powder was spark plasma sintered to obtain pellets with 97 % relative density. The XRD pattern of the as-sintered pellet (fig.3) is fully indexed with the skutterudite structure type. Rietveld refinement results in a lattice parameter a = 9.0527(2) Å which is significantly larger than that of the as-synthesized powder. Along with the disappearance of the InSb secondary phase, whose melting point (789 K) is lower than the sintering temperature, this suggests that InSb reacts with the skutterudite matrix during the sintering step thus increasing the inserted In content. According to literature data, this lattice parameter corresponds to a true In x Co 4 Sb 12 composition close to x = 0.20 -0.22[START_REF] Tang | Phase diagram of In-Co-Sb system and thermoelectric properties of In-containing skutterudites[END_REF][START_REF] Grytsiv | In y Co 4 Sb 12 skutterudite: phase equilibria and crystal structure[END_REF]. The scenario is very similar to the synthesis of In 0.13 Co 4 Sb 12 where about 20 % of In was inserted in the structure during the sintering step[START_REF] Le Tonquesse | Innovative one-step synthesis of mesostructured CoSb 3 -based skutterudites by magnesioreduction[END_REF].

Figure 4 :

 4 Figure 4: (a) Secondary electron SEM image of a broken cross-section of a densified In 0.22 Co 4 Sb 12 pellet and histogram showing the distribution of the apparent grain size. (b) Backscattered electron SEM image and corresponding EDS elementary mappings of the pellet polished surface.

Figure 5 :

 5 Figure 5: High-temperature dependence of (a) the electrical resistivity, (b) Seebeck coefficient, (c) power factor, (d) total (squares) and lattice (crosses) thermal conductivities and (e) figure-of-merit of In 0.22 Co 4 Sb 12 synthesized by magnesioreduction (filled black squares) along with literature data for In 0.25 Co 4 Sb 12 taken from [11] (green empty squares) and In 0.28 Co 4 Sb 12 taken from [2] (red empty squares).

7 10 - 8 W

 8 Ω K -2 . The κ L of the magnesiosynthesized sample agrees with the lower κ L values of the literature over the entire temperature range. No significant decrease of κ L is measured at room temperature contrary to our previous work on In 0.13 Co 4 Sb 12 certainlybecause of the larger average grain size, 1.8 µm vs. 600 nm, respectively, and the stronger influence of the rattlers over the mesostructure at such elevated concentration. As a consequence of the lower PF, a ZT max of 0.95 at 750 K is obtained which remains 5 and 15 % lower than the reference samples made by conventional melting/annealing process.

  thesis of In-filled skutterudite from Sb 2 O 4 and In-doped Co 3 O 4 evidenced a complex scenario involving intermediate species: CoO, Sb 2 O 3 , CoSb 2 O 6 and CoSb 2 O 4 . The formation of CoSb 3 precedes the insertion of In-rattler in the cage. After spark plasma sintering, the resulting material is single phase skutterudite with composition close to In 0.22 Co 4 Sb 12 which corresponds to the In-richest composition which could be synthesized by this technique in these conditions. A ZT max of 0.95 is measured at 750 K due to limited PF resulting from elevated ρ caused by microcracks appearing in the pellets. In addition to im-portant energy and time saving, the relatively mild reaction conditions used in this process prevent high Mg vapors pressure inside the reactor thus avoiding the formation of deleterious Mg-containing side-products which often limits the up-scaling perspectives of magnesioreduction processes. Finally, the knowledge gained on the reaction mechanism will be a precious help for the development of optimized reaction conditions (multi-step heat treatment, mixed oxide precursors) enabling the insertion of other filler atoms (e.g. Ba, rare earths elements) whose respective oxides are often too stable to be reduced by Mg in the present reaction conditions.

  2 O 6 [Co 2+ , Sb 5+ ] (P4 2 /mnm) and CoSb 2 O 4 [Co 2+ , Sb 3+ ] (P4 2 /mbc) ternary intermediates are formed most likely by solid state reactions between the various binary oxides in presence. This is supported by several works reporting the synthesis of these mixed oxides by conventional solid-state reaction starting from CoO and Sb 2 O 3 /Sb 2 O 5 powders between 973 and 1073 K [30, 31, 32]. At this point of the reaction, Sb 5+ and Co 3+ are no more present in the reaction media and Sb 3+ and Co 2+ are consecutively reduced in metallic Co and Sb. The complete reduction of CoSb 2 O 4 and Sb 2 O 3 into CoSb 3 takes another 36 2+ can substitute Sb to a great extent in CoSb 2 O 4 [30]. In presence of InSb, the lattice parameter of In x CoSb 3 increases from 9.0384(2) Å after 48 h to 9.04872(4) Å after 96 h. This can be explained by the insertion of In in the CoSb 3 structure according to: Co 4 Sb 12 + xInSb --→ In x Co 4 Sb 12 + xSb

	Traces of InSb appear on the diffraction patterns after 72 h. We hypothesize
	that In was solubilized in CoSb 2 O 4 because (i) the formation of InSb corre-
	sponds to the total reduction of CoSb 2 O 4 , (ii) the refined lattice parameters of
	the latter, a = 8.5078(5) Å and c = 5.9316(5) Å, are significantly larger than
	literature data, a = 8.49285(7) Å and c = 5.92449(5) Å [30] and (iii) other ele-
	ments such as Pb
	hours according to:
	2 CoSb 2 O 4 + Sb 2 O 3 + 11Mg --→ 2 CoSb 3 + 11MgO	(6)

After 36 h, no trace of CoSb 2 O 6 remains in the XRD pattern (fig. 3) and CoSb 2 O 4 is the major phase representing 60 mol.% of the sample. According to the simultaneous decrease of CoSb 2 O 6 and increase of CoSb 2 O 4 contents, one might expect CoSb 2 O 4 to be formed from the reduction of CoSb 2 O 6 according to: CoSb 2 O 6 + 2 Mg --→ CoSb 2 O 4 + 2 MgO (5) More interestingly, skutterudite starts forming (≈ 10 %) and the lattice parameter a = 9.0384(8) Å corresponds well to In x Co 4 Sb 12 with low In-content (x < 0.05) [7, 8].

  to 0.22 in the MR-samples decreases the thermal conductivity and increases the ZT max from 0.75 to 0.95. Oversaturating In in CoSb 3 induces the formation of InSb and/or CoSb 2 (nano)precipitates that are playing a significant role in decreasing the thermal conductivity and improving the ZT max of these composite materials [37, 38, 39, 40]. MR-In 0.22 Co 4 Sb 12 do not show the presence of such precipitates after spark plasma sintering. The measured TE properties can thus be considered as intrinsic to the material and slightly deteriorated by the microcracks appearing in the pellets during physical properties measurements. The submicron particle size distribution obtained after magnesioreduction corresponds well to that reported by Benyahia et al. on mesostructured ZT = 1.4 In 0.25 Co 4 Sb 12 . Unfortunately, the grain growth induced during sintering cancels this microstructural feature and both thermal conductivity and figure-of-merit are much closer from those obtained by fusion-

solidification-long term annealing

Acknowledgements

Loic Joanny and Francis Gouttefangeas are acknowledged for SEM images and EDS analyses performed on the CMEBA platform belonging to the ScanMAT unit (UMS 2001, University of Rennes 1) which received a financial support from the European Union (CPER-FEDER 2007-2014).

Declaration of interest

The authors declare no conflict of interest.


Bettoni Jérémie
email: jeremiebettoni@gmail.com

Olivetto Matthieu

Bouaoud Jébrane

Devauchelle Bernard

M.D Jérémie Bettoni

Technical note on the harvest of periosteal forearm composite free flaps in the 2 treatment of early mandibular osteoradionecrotic injury 3 4 5

Keywords: Osteoradionecrosis, periosteal free flap, management, revascularization surgery, 44 forearm periosteal free flap 45

25 26 Periosteal radial forearm free flaps allow functional and curative treatment of 27 osteoradionecrotic jaw lesions. The flap responds physiologically to hypoxia, 28 hypocellularity, and hypovascularity, which are characteristic results of 29 osteoradionecrotic injury. Here, we propose a reproducible, simple, and safe 30 method for harvesting a forearm free flap, allowing us to obtain a periosteal 31 composite free flap. Our technique offers potential early management of 32 osteoradionecrotic lesions resistant to medical treatment. Furthermore, the 33 forearm periosteal composite free flap offers the advantage of a long vascular 34 pedicle which is ideally suited for necks which have been submitted to 35 irradiation or multiple operations. Thusly, vascular anastomosis can be 36 performed at a distance from the irradiated areas. Compared to other periosteal 37 free flaps, such as those harvested from the iliac crest or the internal femoral 38 condyle, the presence of a skin paddle facilitates clinical flap monitoring, 39 optimizes the restoration of bone sealing, and facilitates the treatment of 40 scarring.

Introduction

with an efficacy rate of between 86% and 100%. [START_REF] Lee | Outcomes of microvascular free flap reconstruction 170 for mandibular osteoradionecrosis : A systematic review[END_REF][START_REF] Bettoni | Role of « revascularization 174 surgery » in the management of mandibular osteoradionecrosis lesions : a report of 21 years of 175 experience[END_REF] By contrast, for treatment 56 requiring a consensus, such as long-term antibiotic therapy or the Pentoclo protocol, 57 efficacy assessment requires evaluation by randomized double-blind placebo-58 controlled studies. [START_REF] Martos-Fernandez | Pentoxifulline, Tocopherol, and Clodronate for the treatment of 179 mandibular osteoradionecrosis: a systematic review[END_REF] However, due to the difficulties of surgery in irradiated areas, the 59 scarcity of vessels, and the high risk of failure, most teams consider it preferable to 60 reserve radical management with free flap reconstruction for cases of ORN with 61 fractures. [START_REF] Rice | The management of 183 osteoradionecrosis of jaws -A review[END_REF] Furthermore, malnutrition secondary to reactional trismus is a criterion of The remaining steps of harvesting are the same as the conventional procedures 105 described by Yang, in 1981. [START_REF] Yang | Forearm free flap transplantation: a 191 report of 56 cases[END_REF] The careful dissection of the radial vascular pedicle is 106 carried out with ligatures of all collaterals until a sufficient vascular pedicle length is 107 obtained to allow for anastomosis (Figure 2). Finally, after releasing the pneumatic

  ORN) is a severe complication of radiotherapy 49 characterized by hypoxic, hypovascular, and hypocellular bone 1 which has lost its 50 capacity to renew itself. Clinically, ORN presents as spontaneous mandibular bone 51 exposure or occurs following an endobuccal surgical procedure. 2 Different 52 complications can occur, in particular cutaneous fistulas and/or pathological 53 fractures. Therapeutic revascularization surgery (resection of the pathological bone 54 with immediate reconstruction by microanastomosis of a flap) is the gold standard, 55

  62 severity, complicating any surgical management. In 2009, D'Hauthuille et al. 7 63 proposed the early management of ORN relating to the underlying risk of neoplasia 64 and the tendency of the lesions to progress. This management is based on marginal 65 bone resection with reconstruction by a free flap "cover" which preserves the food 66 masticating function while revascularizing the underlying bone. Thus, periosteal free 67 flaps (internal condyle, femoral, or others) are recommended for their osteoinductive 68 and osteogenic properties. Indeed, the supply of vascularized living tissues within an 69 irradiated environment constitutes a physiological response to the treatment of 70 osteoradionecrosis. The efficacy rate of 100% confirms that it remains essential to 71 design mandibular ORN curative surgery with a unique and functional approach.

72

  From this perspective, and taking into account the scarring fibrosis of the post-radial 73 endobuccal mucosa, the forearm free flap with a radial periosteal component offers 74 several advantages responding to the constraints of early osteoradionecrotic lesion 75 revascularization surgery: i) it provides a periosteal membrane, and ii) its 76 fasciocutaneous flap facilitates clinical flap monitoring and optimizes buccal 77 tightness with tension-free sutures. Here, we describe our harvesting technique, 78 ensuring fast and safe dissection.

  ulnar vascularization of the palmar arch using Allen's test, the 82 dimensions of a fasciocutaneous paddle is defined, centered on the radial vascular 83 pedicle. It is important to overestimate the volume of the cutaneous paddle needed 84 relative to the bone exposure area to allow tension-free suturing during endobuccal 85 insertion. 86 Using a pneumatic tourniquet, a curvilinear incision starting from the fold of the 87 elbow to the upper point of the skin paddle is performed. Then, the ulnar and radial 88 sides of the skin paddle should be carefully incised to include the cephalic vein. It is 89 important to begin the dissection of the fasciocutaneous paddle by the radial side of 90 the flap to protect the cutaneous branches of the radial nerve. Once dissected and 91 reclined by placing a Gillies hook or a Faraboeuf spreader on the brachioradial 92 muscle tendon (Figure1), the radial periosteum is incised with the length 93 corresponding to the slice of mandibular bone section to be "resurfaced". The 94 periosteum is then gently roughened to avoid it dissociating from the radial vascular 95 pedicle.96The ulnar surface of the flap is secondarily dissected up to the tendon of the long 97 flexor muscle of the carpus, which is then retracted with a Gillies hook, allowing 98 exposure of the lower end of the radius. This facilitates the access to the radial 99 periosteum, which should be incised and scraped until it reaches the contralateral 100 detachment zone. Once the entire periosteum is released, the distal end of the radial 101 vascular pedicle is dissected, then ligated to counter-incise the radial periosteum and 102 release it over its entire length. The limit of the proximal portion of the radial 103 periosteum is defined according to the required length. 104

Figure 1 :Figure 2 :

 12 Figure 1: Intraoperative photograph of the harvesting of the radial periosteal paddle (black arrow) 224

Acknowledgements 159

Text

Conflict of Interest Statement: None 161


Sayani Das

Asuncion Carmona

Kaustav Khatua

Francesco Porcaro

Andrea Somogyi

Richard Ortega

Ankona Datta
email: ankona@tifr.res.in

Manganese Mapping Using a Fluorescent Mn 2+ Sensor and Nanosynchrotron X-ray Fluorescence Reveals the Role of the Golgi Apparatus as a Manganese Storage Site

Keywords: Manganese sensing, Golgi apparatus, fluorescent manganese sensors, synchrotron X-ray fluorescence nano-imaging

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

Manganese Mapping Using a Fluorescent Mn 2+

INTRODUCTION

The interplay between metal ion compartmentalization and dynamics has emerged as a prominent concept in investigations into the regulatory and signaling roles of transition metal ions. [START_REF] Hong-Hermesdorf | Subcellular metal imaging identifies dynamic sites of Cu accumulation in Chlamydomonas[END_REF][START_REF] Chang | Bioinorganic Life and Neural Activity: Toward a Chemistry of Consciousness?[END_REF][START_REF] Que | Quantitative mapping of zinc fluxes in the mammalian egg reveals the origin of fertilization-induced zinc sparks[END_REF] Existing challenges in metallo-biology are related to addressing critical questions in metal ion homeostasis some of which include: How are correct metal ions installed in protein active sites? [START_REF] Waldron | How do bacterial cells ensure that metalloproteins get the correct metal?[END_REF] How do biological systems regulate metal ion mobilization in response to external conditions? [START_REF] Hong-Hermesdorf | Subcellular metal imaging identifies dynamic sites of Cu accumulation in Chlamydomonas[END_REF] Can we identify threshold levels for competing pathways of metal ion channeling especially what concentrations trigger pathological pathways? [START_REF] Mukhopadhyay | Manganese Blocks Intracellular Trafficking of Shiga Toxin and Protects Against Shiga Toxicosis[END_REF][START_REF] Mukhopadhyay | Shiga toxin-binding site for host cell receptor GPP130 reveals unexpected divergence in toxin-trafficking mechanisms[END_REF] Which pathways are vulnerable to pathogens and how do pathogens hack the cellular metal ion inventory? [START_REF] Brophy | Manganese and microbial pathogenesis: sequestration by the Mammalian immune system and utilization by microorganisms[END_REF][START_REF] Zygiel | Transition Metal Sequestration by the Host-Defense Protein Calprotectin[END_REF] Multiple recent reports have tried to answer these questions. [START_REF] Hong-Hermesdorf | Subcellular metal imaging identifies dynamic sites of Cu accumulation in Chlamydomonas[END_REF][START_REF] Chang | Bioinorganic Life and Neural Activity: Toward a Chemistry of Consciousness?[END_REF][START_REF] Que | Quantitative mapping of zinc fluxes in the mammalian egg reveals the origin of fertilization-induced zinc sparks[END_REF] Results from these studies highlight the ability of biological systems to optimally use compartmentalization and dynamically re-purpose compartments in response to external and internal stimuli. [START_REF] Hong-Hermesdorf | Subcellular metal imaging identifies dynamic sites of Cu accumulation in Chlamydomonas[END_REF][START_REF] Chang | Bioinorganic Life and Neural Activity: Toward a Chemistry of Consciousness?[END_REF][START_REF] Que | Quantitative mapping of zinc fluxes in the mammalian egg reveals the origin of fertilization-induced zinc sparks[END_REF] In this context, strategic combination of molecular biology tools, chemical probes, and metal ion imaging techniques have led to the discovery of new transporters, compartments, and pathways in metal ion homeostasis.

Further, these efforts have brought forth previously unanticipated roles of transition metal ions like copper, iron, and manganese in biological decision making and defense processes. [START_REF] Barnese | Manganous Phosphate Acts as a Superoxide Dismutase[END_REF][START_REF] Dodani | Copper is an endogenous modulator of neural circuit spontaneous activity[END_REF][START_REF] Carter | Fluorescent Sensors for Measuring Metal Ions in Living Systems[END_REF][START_REF] Que | Metals in neurobiology: probing their chemistry and biology with molecular imaging[END_REF][START_REF] Cotruvo | Synthetic fluorescent probes for studying copper in biological systems[END_REF][START_REF] Das | Emerging chemical tools and techniques for tracking biological manganese[END_REF][START_REF] Da Cunha | Overview of chemical imaging methods to address biological questions[END_REF] Manganese has recently been in the focus due to reports on novel roles of the labile manganese pool and identification of manganese specific transporters. [START_REF] Barnese | Manganous Phosphate Acts as a Superoxide Dismutase[END_REF][START_REF] Daly | Accumulation of Mn(II) in Deinococcus radiodurans facilitates gamma-radiation resistance[END_REF][START_REF] Mcnaughton | Probing in vivo Mn 2+ speciation and oxidative stress resistance in yeast cells with electron-nuclear double resonance spectroscopy[END_REF][START_REF] Sharma | Responses of Mn2+ speciation in Deinococcus radiodurans and Escherichia coli to gammaradiation by advanced paramagnetic resonance methods[END_REF][START_REF] Tuschl | Mutations in SLC39A14 disrupt manganese homeostasis and cause childhood-onset parkinsonism-dystonia[END_REF][START_REF] Sharma | Across the tree of life, radiation resistance is governed by antioxidant Mn 2+ , gauged by paramagnetic resonance[END_REF] In 2004, Daly and coworkers provided evidence that related the high-radiation resistance of bacteria like Deinococcus radiodurans to Mn 2+ accumulation in this species in a concentration dependent manner. [START_REF] Daly | Accumulation of Mn(II) in Deinococcus radiodurans facilitates gamma-radiation resistance[END_REF] The result led to a series of studies aimed toward identifying the Mn 2+ species responsible for radiation resistance. These studies revealed the importance of low molecular weight labile Mn 2+ species in conferring radiation resistance and opened up the hypothesis that labile Mn 2+ species might act as anti-oxidants. [START_REF] Daly | Accumulation of Mn(II) in Deinococcus radiodurans facilitates gamma-radiation resistance[END_REF] While the superoxide dismutase activity of labile Mn 2+ had been proposed earlier, Valentine and coworkers showed in 2012 that labile Mn 2+ complexes could catalyze superoxide dismutation under physiological concentrations. [START_REF] Barnese | Biologically relevant mechanism for catalytic superoxide removal by simple manganese compounds[END_REF] These series of studies pointed to a significant biological role of the labile Mn 2+ pool other than providing metal ions for protein-binding. At about the same time, in 2012, a Zn 2+ transporter, ZnT10, was reclassified as an exclusive Mn 2+ transporter, SLC30A10. [START_REF] Tuschl | Syndrome of hepatic cirrhosis, dystonia, polycythemia, and hypermanganesemia caused by mutations in SLC30A10, a manganese transporter in man[END_REF] A set of Mn 2+ transporters which included SLC30A14, SLC39A14 and SLC39A8 were also identified. [START_REF] Tuschl | Mutations in SLC39A14 disrupt manganese homeostasis and cause childhood-onset parkinsonism-dystonia[END_REF][START_REF] Jenkitkasemwong | Physiologic implications of metal-ion transport by ZIP14 and ZIP8[END_REF][START_REF] Park | SLC39A8 Deficiency: A Disorder of Manganese Transport and Glycosylation[END_REF][START_REF] Xin | Manganese transporter Slc39a14 deficiency revealed its key role in maintaining manganese homeostasis in mice[END_REF][START_REF] Saudubray | An overview of inborn errors of metabolism affecting the brain: from neurodevelopment to neurodegenerative disorders[END_REF] Further, between 2009 and 2016, multiple studies by Tuschl and co-workers showed that mutations in the transporters, SLC39A14 and SLC30A10, led to manganese induced Parkinsonism in young adults and children. [START_REF] Tuschl | Mutations in SLC39A14 disrupt manganese homeostasis and cause childhood-onset parkinsonism-dystonia[END_REF][START_REF] Tuschl | Syndrome of hepatic cirrhosis, dystonia, polycythemia, and hypermanganesemia caused by mutations in SLC30A10, a manganese transporter in man[END_REF][START_REF] Stamelou | Dystonia with brain manganese accumulation resulting from SLC30A10 mutations: a new treatable disorder[END_REF][START_REF] Mukhtiar | Hypermanganesemia with Dystonia, Polycythemia and Cirrhosis (HMDPC) due to mutation in the SLC30A10 gene[END_REF][START_REF] Mukhopadhyay | Familial manganese-induced neurotoxicity due to mutations in SLC30A10 or SLC39A14[END_REF] The past two decades have been extremely prolific with studies on Mn, identifying new roles of the labile Mn 2+ pool both in physiological and pathophysiological states.

Importantly, these studies have led to a niche area in Mn biology which is directly trying to address Mn 2+ localization and dynamics along with the localization of Mn 2+ transporters in the context of these new discoveries.

The identification of intra-cellular locations of Mn 2+ pools initially relied on experiments involving uptake of 54 Mn 2+ and organelle isolation to determine the extent of uptake within an organelle under a specific condition. [START_REF] Mukhopadhyay | Identification of a gain-of-function mutation in a Golgi P-type ATPase that enhances Mn2+ efflux and protects against toxicity[END_REF] Mukhopadhyay and Linstedt showed in 2011 that the introduction of a gain-of-function mutation in SPCA1 which is a Golgi localized Mn 2+ influx transporter increased Mn 2+ levels in the Golgi and protected cells against Mn 2+ toxicity. [START_REF] Mukhopadhyay | Identification of a gain-of-function mutation in a Golgi P-type ATPase that enhances Mn2+ efflux and protects against toxicity[END_REF] Importantly, cells expressing the mutant SPCA1 transporter showed 70% increased cell viability upon exposure to mM levels of Mn 2+ when compared to wild-type cells indicating a plausible detoxification role of the Golgi. One of the functions of the Golgi apparatus involves metal ion sequestration into the lumen. [START_REF] Sengupta | Control of organelle size: the Golgi complex[END_REF] The sequestered metal ions are either channeled into the cytoplasm for metal ion related functions or putatively removed into the extra-cellular space under metal overload conditions. [START_REF] Sengupta | Control of organelle size: the Golgi complex[END_REF] The localization of Mn within the Golgi under Mn overexposure conditions was also revealed by Carmona and co-workers in 2010. [START_REF] Carmona | Manganese Accumulates within Golgi Apparatus in Dopaminergic Cells as Revealed by Synchrotron X-ray Fluorescence Nanoimaging[END_REF] Synchrotron X-ray fluorescence (SXRF) nano-imaging was used to generate Mn elemental maps in frozen cells and showed Mn localization in the Golgi apparatus in rat dopaminergic cells. [START_REF] Carmona | Manganese Accumulates within Golgi Apparatus in Dopaminergic Cells as Revealed by Synchrotron X-ray Fluorescence Nanoimaging[END_REF][START_REF] Ortega | Golgi apparatus functions in manganese homeostasis and detoxification[END_REF] This result was obtained by developing a correlative microscopy approach combining SXRF imaging and organelle fluorescence microscopy. [START_REF] Carmona | Manganese Accumulates within Golgi Apparatus in Dopaminergic Cells as Revealed by Synchrotron X-ray Fluorescence Nanoimaging[END_REF][START_REF] Roudeau | Correlative organelle fluorescence microscopy and synchrotron X-ray chemical element imaging in single cells[END_REF] Following this work, multiple studies on different cell types showed clear Mn localization in the Golgi apparatus under different Mn exposure conditions (Table S1, ESI †). A point to note from these reports was that the cells used for these studies which included both neuronal cells and HeLa cells, did not express the Mn 2+ efflux transporter SLC30A10. Mutations in this transporter are directly related to childhood onset of manganese induced Parkinsonism. [START_REF] Carmona | SLC30A10 Mutation Involved in Parkinsonism Results in Manganese Accumulation within Nanovesicles of the Golgi Apparatus[END_REF][START_REF] Leyva-Illades | SLC30A10 Is a Cell Surface-Localized Manganese Efflux Transporter, and Parkinsonism-Causing Mutations Block Its Intracellular Trafficking and Efflux Activity[END_REF] Further, Carmona and co-workers showed that overexpression of SLC30A10 in HeLa cells led to a loss of Mn signal from the Golgi apparatus in SXRF Mn elemental maps. [START_REF] Carmona | SLC30A10 Mutation Involved in Parkinsonism Results in Manganese Accumulation within Nanovesicles of the Golgi Apparatus[END_REF] These results highlighted the relevance of the Golgi apparatus as a compartment for channeling excess Mn under Mn overload and dysregulation conditions. With the identification of the Golgi apparatus as a possible detoxification compartment for excess Mn ions, it becomes imperative to evaluate possibilities of employing imaging techniques that can be applied to living samples, like confocal microscopy in conjunction with Mn 2+ selective fluorescent sensors to tease apart the dynamics of the Mn 2+ labile pool with respect to the Golgi apparatus. Hence, in this paper we evaluate a sensitive fluorescent Mn 2+ sensor for its ability to locate manganese in the Golgi within HEK293T cells under sub-cytotoxic levels of manganese exposure. We compare the results from confocal microscopy using the Mn 2+ sensor with nano-SXRF imaging under identical conditions to show that Mn localizes within the Golgi apparatus in these cells under sub-cytotoxic Mn exposure conditions. Synthesis of the manganese sensor M1. The M1 sensor was synthesized and purified according to a previously reported procedure (general procedures and materials listed in previous section). [START_REF] Bakthavatsalam | Tuning macrocycles to design 'turn-on' fluorescence probes for manganese(ii) sensing in live cells[END_REF] Cell culture for confocal imaging. HEK 293T cells were cultured in Dulbecco's Modified Eagle's Medium (DMEM, Sigma-Aldrich ® ) supplemented with Fetal Bovine Serum (10%, Gibco ® ), Penicillin (50 units/ml, Gibco ® ) and Streptomycin (50 μg/ml, Gibco ® ) in T25 culture flasks at 37 °C under humidified air containing 5% CO 2 . A day before the imaging, the cells were plated on glass coverslip bottomed petri plates (35 mm diameter, Tarsons) coated with polylysine (0.1 mg/mL) and fibronectin (100 µg/mL).

EXPERIMENTAL SECTION

METHODS

General

All microscopy experiments were carried out at 25 o C using an LSM 880 confocal microscope (Zeiss) and images were collected in the internal detector channels using a 40X water immersion objective (Zeiss). Z-series were obtained with a spacing of 0. Cell culture for SXRF imaging. For synchrotron X-ray fluorescence imaging, cells were cultured directly on sample holders specifically adapted to the synchrotron beamline using protocols established at CENBG. [START_REF] Perrin | Evaluation of sample preparation methods for single cell quantitative elemental imaging using proton or synchrotron radiation focused beams[END_REF] The sample holders made of PEEK had a hole in the middle where a 2 µm high grade polycarbonate film was stretched over and glued. The polycarbonate film was treated with gelatine gel (2% in water) to allow cell adhesion. [START_REF] Roudeau | Correlative organelle fluorescence microscopy and synchrotron X-ray chemical element imaging in single cells[END_REF][START_REF] Carmona | SLC30A10 Mutation Involved in Parkinsonism Results in Manganese Accumulation within Nanovesicles of the Golgi Apparatus[END_REF] Cells were seeded at low-density 10,000 cell/cm 2 . Two days after plating, cells were transduced with CellLight Golgi-RFP. Cells were treated with 2 µL of BacMam reagent per 10,000 cells for 20 hours. After this time, the medium was removed and cells were exposed for 4 h at the desired concentration of MnCl 2 (25 µM and 250 μM). Then the culture medium was removed and cells were exposed to 10 µg/mL Hoechst 33342 nuclear dye solution for 15 min in darkness and at 37 °C. Afterwards, fluorescence images of living cells were acquired using an epifluorescence microscope (Olympus BX51, Tokyo, Japan) and using the set of filters U-MWU2 BP330-385 (Excitation) BA420 (Emission) for blue emission, and U-MWIG2 BP520-550 (Excitation) BA580IF

(Emission) for red emission.

After epifluorescence microscopy observation, in order to analyze the constitutive elements of the cells by SXRF, samples must be cryofixed and freeze dried to preserve the cellular structure and elemental content. [START_REF] Roudeau | Correlative organelle fluorescence microscopy and synchrotron X-ray chemical element imaging in single cells[END_REF][START_REF] Perrin | Evaluation of sample preparation methods for single cell quantitative elemental imaging using proton or synchrotron radiation focused beams[END_REF] For this purpose, the samples were quickly washed first with PBS and second with ultrapure water, and rapidly plunge-frozen for 20 sec into isopentane and cooled at -164°C with liquid nitrogen. After cryofixation, remaining traces of isopentane were blotted with a paper filter and samples were stored in liquid nitrogen vapors until freeze-drying. SXRF analyses at room temperature are incompatible with the presence of liquid water, so the samples were freeze-dried at -85 °C under vacuum (2.5 x 10 -3 mbar) using Christ alpha 2-4 LD plus freeze-dryer, for 72 hours.

SXRF imaging. Nano-SXRF imaging was performed at the NANOSCOPIUM beamline [START_REF] Somogyi | Optical design and multi-length-scale scanning spectro-microscopy possibilities at the Nanoscopium beamline of Synchrotron Soleil[END_REF][START_REF] Hostachy | Graftable SCoMPIs enable the labeling and X-ray fluorescence imaging of proteins[END_REF] , Synchrotron SOLEIL, in France. Analyses were conducted at room temperature on cryofixed and freeze-dried cells. A 10 keV photon beam was focused at 300 x 300 nm 2 in the sample focal plane with a flux of 10 10 ph•s -1 . Elemental maps were measured by the Flyscan continuous sample scanning technique [START_REF] Medjoubi | Development of fast, simultaneous and multitechnique scanning hard X-ray microscopy at Synchrotron Soleil[END_REF] with 300 x 300 nm 2 spatial resolution. Full X-ray spectra were detected in each pixel with 300 ms/pixel exposure time using two Si-SDD detectors located at 120° respectively to beam direction and at both sides of the sample. Intracellular potassium and chloride was used in SXRF imaging to locate the cell position and hence perform element/organelle correlative imaging.

RESULTS and DISCUSSION

Investigating intra-cellular Mn 2+ localization using a fluorescent Mn 2+ sensor Metal ion imaging using a selective fluorescent metal ion sensor in a confocal microscopy set-up can provide direct information on intracellular localization of a specific metal ion. [START_REF] Hong-Hermesdorf | Subcellular metal imaging identifies dynamic sites of Cu accumulation in Chlamydomonas[END_REF][START_REF] Que | Quantitative mapping of zinc fluxes in the mammalian egg reveals the origin of fertilization-induced zinc sparks[END_REF][START_REF] Carter | Fluorescent Sensors for Measuring Metal Ions in Living Systems[END_REF][START_REF] Que | Metals in neurobiology: probing their chemistry and biology with molecular imaging[END_REF][START_REF] Cotruvo | Synthetic fluorescent probes for studying copper in biological systems[END_REF][START_REF] Khan | Two-photon imaging of Zn2+ dynamics in mossy fiber boutons of adult hippocampal slices[END_REF] However, interpretation of imaging data critically depends on prior information on the inherent localization propensities of the sensor. Imaging the sub-cellular localization of Mn 2+ using a fluorescent Mn 2+ sensor has not been achieved till date, firstly, because of the lack of subcellular organelle targeted Mn 2+ sensors. Secondly, there is no clear information on the subcellular targetability of the few existing Mn 2+ sensors. The scarcity of fluorescent Mn 2+ sensors (add references) which arises from coordination chemistry challenges in developing selective

Mn 2+ sensors and unavailability of detailed information on their intra-cellular targetability, severely mars the ability to utilize fluorescence microscopy to localize Mn 2+ within cells.

However, as mentioned in the introduction, recent developments in Mn 2+ biology especially related to dynamics in Mn 2+ localization under different external and internal conditions necessitate a two-pronged approach: 1. The development of new organelle targeted Mn 2+ sensors and 2. Investigations on existing Mn 2+ sensors to check if these sensors can be strategically used to gain information on the sub-cellular localization of Mn 2+ ions. In this paper we focus on the later approach.

Scheme 1. 'Turn-on' Mn 2+ sensor, M1, [START_REF] Bakthavatsalam | Tuning macrocycles to design 'turn-on' fluorescence probes for manganese(ii) sensing in live cells[END_REF] used for imaging Mn 2+ localization in living cells in this work.

In order to investigate the intracellular Mn 2+ localization in live cells, we decided to use an existing fluorescent Mn 2+ sensor, M1, that had been previously reported in our laboratory. M1 is a cell-permeable 'turn-on' fluorescent Mn 2+ sensor (Scheme 1). [START_REF] Bakthavatsalam | Tuning macrocycles to design 'turn-on' fluorescence probes for manganese(ii) sensing in live cells[END_REF] While the sensor has limited aqueous solubility, in an earlier report we had shown the molecule could still be applied successfully for live cell imaging of Mn 2+ in HEK293T cells under low levels of sub-cytotoxic (25 µM) external Mn 2+ exposure by preparing samples in 1 % DMSO in buffer. [START_REF] Bakthavatsalam | Tuning macrocycles to design 'turn-on' fluorescence probes for manganese(ii) sensing in live cells[END_REF] The sensor had revealed peri-nuclear punctate staining in cells and we had attributed the punctate staining to the ability of the sensor to localize in lipid rich regions and detect Mn 2+ in those compartments. [START_REF] Bakthavatsalam | Tuning macrocycles to design 'turn-on' fluorescence probes for manganese(ii) sensing in live cells[END_REF] The ability of the sensor to detect Mn 2+ within cells had been confirmed by competition studies with a cell-permeable divalent metal ion chelator. However, we had not further investigated the identity of the compartments in which the sensor was detecting Mn 2+ ions. As there has been mounting evidence on the intra-cellular localization of Mn 2+ in the Golgi apparatus under Mn 2+ exposure conditions, and we had earlier observed punctate staining near the peri-nuclear region with our Mn 2+ sensor it became necessary to carefully check if a sub-set of the compartments that were showing high fluorescence intensity was the Golgi apparatus and if the sensors could be used to gain further information on Mn 2+ localization in living cells.

We therefore used the existing M1 sensor and checked if the fluorescence intensity in HEK293T cells correlated with the location of the Golgi apparatus in these cells. Three sets of experiments were performed on live cells with: 1. no external Mn exposure; 2. exposure to 25 µM MnCl 2 ; and 3. exposure to 250 µM MnCl 2 . In order to identify which intra-cellular compartments exhibited high fluorescence intensity in the presence of the sensor, the cells were incubated with different organelle trackers that could mark the Golgi apparatus, endosomes, lysosomes, and mitochondria (Figures 1, S1, S2, and S3). As the M1 sensor emitted in the green region of the spectrum (λ ex 488 nm / λ em 495-536 nm), red-emitting organelle trackers (λ ex 543 nm / λ em 570-680 nm) were chosen for the experiments to ensure minimal overlap between the emission of the Mn 2+ sensor with that of the organelle trackers. Hence, both Mn untreated and treated HEK293T cells were incubated with the M1 sensor and an organelle tracker and fluorescence images of the cells were recorded on a confocal microscope. In the case of the Golgi tracker a red-fluorescent protein (RFP) tagged marker was transduced into the cells prior to the experiments. The rest of the trackers could be applied via incubation. Importantly, in order to ensure that the co-localization data was interpreted accurately we performed control confocal imaging experiments to check whether there was any fluorescence bleed through from the M1 sensor to the organelle trackers and vice versa. We observed that there was no emission in the green channel when the organelle trackers were excited. However, there was some emission in the red channel for the endosome, lysosome, and Golgi trackers when the M1 sensor was excited. This could be attributed to the tail of the emission spectra of the M1 sensor (Figure S4A) which ranges from 565 nm to 600 nm.

Although this would be a minor contribution, we corrected the M1 sensor-organelle colocalization imaging data for bleed through. S3). The thresholded Mander's split colocalization coefficients for cells exposed to 250 µM Mn 2+ were 0.8 and 0.7 for the Golgi tracker and M1 sensor channels, respectively indicating that the M1 sensor fluorescence clearly colocalized with the Golgi tracker. Importantly, intensity for the M1 sensor emission channel was also observed in the perinuclear regions even in cells that had not been externally exposed to Mn 2+ . Visible inspection of the co-localized images (Figure 1Ad) showed that the intensity of the Golgi tracker remained constant while the intensity of the M1 sensor increased with increasing levels of Mn exposure as indicated by the regions pointed with white arrows which show a progressive color change from orange to yellow. Since M1 is a 'turn-on' Mn 2+ sensor (Figure S4B) this data indicated that Mn 2+ ions were present in the Golgi apparatus of HEK293T cells and the concentration Mn 2+ ions increased with increasing levels of external Mn 2+ exposure.

In order to confirm that the increase in emission of the M1 sensor within the Golgi apparatus was indeed due to the presence of higher levels of Mn 2+ in the Golgi apparatus of cells exposed to Mn 2+ , we performed several control experiments. As M1 is a photo-induced electron transfer based sensor, protonation of the amine group of the dimethylaniline moiety in the sensor scaffold (Scheme 1) might lead to a 'turn-on' response. The pH of the Golgi apparatus is slightly acidic and is maintained in the pH range of 6.4 to 6.8. [START_REF] Llopis | Measurement of cytosolic, mitochondrial, and Golgi pH in single living cells with green fluorescent proteins[END_REF] Hence, it was necessary to check if the fluorescence emission of M1 remained constant within the pH range of 6.5 to 7.5. The data indicated minimal change in the fluorescence emission of the sensor between pH 6.0 and pH 8.0 (Figure S5). Further, since our sensor had limited water-solubility, we also checked if there was any effect of solvent polarity on the fluorescence emission of the sensor. We observed a 1.6 times increase in emission intensity for the M1 sensor in ethyl acetate ( = 6.02) with respect to the fluorescence intensity of the sensor in buffer (Figure S6). A 2.2 times enhancement in was observed when the sensor was dissolved in methanol ( = 32.7) in comparison to sensor in buffer (Figure S6). For comparison, the dielectric constant of phospholipid membranes is close to 3.

(add reference) Therefore we might expect a change in the fluorescence emission of the M1 sensor in lipid rich regions similar to that observed for ethyl acetate. However, in comparison to the fluorescence intensity increase that was obtained for the M1 sensor in the presence of Mn 2+ ions (Figure S4), the effect of the polarity of the medium on the fluorescence of the sensor was lower. With these control experiments we can conclude that the 'turn-on' response for the M1 sensor observed in cells treated with Mn 2+ salts can be majorly attributed to the increase in Mn levels.

We next treated the Mn 2+ exposed cells with a cell permeable divalent metal ion chelator tetrakis-(2-pyridylmethyl)ethylene-diamine (TPEN). A decrease in intensity of the M1 sensor channel was observed upon TPEN treatment (Figure 1Aa, rightmost panel). Cells that had not been externally treated with Mn 2+ also showed a visible decrease in intensity in the M1 sensor emission when treated with TPEN indicating that the sensor could detect labile Mn 2+ ions under physiological conditions (Figure S7). Importantly, intensity analysis on confocal images of Mn 2+ untreated cells and Mn 2+ treated cells on selected regions of interest that showed co-localization of the M1 sensor channel with the Golgi tracker, showed a clear increase with increasing levels of Mn 2+ exposure (Figure 1B). Further, TPEN treated cells showed lower intensity in the green channel (Figure 1B). The distinct increase in the emission intensity of the M1 sensor in regions marked with the Golgi tracker with increasing levels of Mn 2+ exposure and decrease in intensity upon TPEN treatment demonstrate that the Golgi apparatus is involved in both channeling and storage of Mn 2+ ions. Further, the presence of Mn 2+ within the Golgi apparatus of cells that were not externally treated with Mn 2+ as indicated by the co-localization of the fluorescence intensity of the M1 sensor in regions marked by the Golgi tracker (Figure 1A, leftmost panels) hints toward a possible role of the Golgi apparatus as an Mn 2+ storage site under physiological conditions. This point can be confirmed in the future with the development second generation organelle targeted Mn 2+ sensors with increased sensitivity. Finally, the fluorescence studies with the M1 sensor indicated that this chemical tool could be applied for studying Mn 2+ regulation within the Golgi apparatus in the future. However, before applying any fluorescent sensor for elucidating mechanistic pathways it would be crucial to cross-validate the results obtained using an orthogonal imaging technique. We therefore chose nano-SXRF imaging as the technique to confirm the observations from our fluorescence microscopy results.

Validating confocal fluorescence imaging results with nano-SXRF imaging

Nano-SXRF imaging provides high resolution elemental maps with 30-300 nm spatial resolution. [START_REF] Somogyi | Optical design and multi-length-scale scanning spectro-microscopy possibilities at the Nanoscopium beamline of Synchrotron Soleil[END_REF] This technique is based on the use of X-ray beams to eject core electrons from atoms and record the atomic X-ray fluorescence emission as electrons from higher energy levels fill in the hole generated in the core shell. SXRF can afford precise elemental maps thus providing a wealth of information on metal ion accumulation in frozen or freeze-dried cells. [START_REF] Zhang | X-ray fluorescence imaging of metals and metalloids in biological systems[END_REF] Nano-SXRF can detect the total metal pool in cells, which includes both metal ions tightly bound to biomolecules and the more labile and chelatable metal pool. Further, the development of protocols that can afford both epifluorescence microscopy and nano-SXRF imaging data on the same biological sample makes the technique applicable for determining organelle specific localization of metal ions. In order to confirm whether Mn 2+ ions indeed accumulate in the Golgi apparatus in HEK293T cells, as indicated by our confocal fluorescence microscopy experiments with the M1 sensor, we performed nano-SXRF imaging studies on the same cell line under identical Mn 2+ exposure conditions. The cells were first transduced with the Golgi-RFP marker followed by exogenous Mn 2+ treatment and incubation with a nucleus staining dye. The living cells were then imaged on an epifluorescence microscopy set-up and later freeze-dried for nano-SXRF imaging. The transmission images of the freeze-dried cells are depicted in Figures 2A and4A and indicate that the cellular morphology was preserved after the sample preparation protocol. The Mn elemental maps generated on cells treated with 25 µM Mn 2+ salts indicated higher intensities in regions (Figure 2C) that co-localized with the Golgi stain in the epifluorescence image (Figure 2B). This result clearly confirmed our fluorescence microscopy observations and indicated that the Golgi apparatus could indeed accumulate Mn 2+ ions in HEK293T cells. The pixel intensities in the Mn maps were low; however the Mn levels observed were close to the detection limit of XRF in a single pixel with 300 ms exposure time. The presence of Mn within the cells was also unambiguously concluded from the sum-spectra of the whole cells (Figure 3). Finally, to revalidate the observation that increased levels of external Mn exposure would lead to increased Mn levels in the Golgi apparatus as shown by our confocal imaging data, we analyzed the nano-SXRF experiments at 250 µM external Mn 2+ exposure level (Figure 4). Representative images of different cells are depicted in Figure 4 and clearly show Mn localization within the Golgi apparatus. Importantly, intensity analysis of the nano-SXRF images from representative samples of the two different sets of experiments (25 µM and 250 µM, respectively) clearly indicated an increase in only the overall cellular Mn levels with higher external Mn 2+ exposure (Figure 3). Further, Mn exposure in these cells did not seem to affect the homeostasis of other essential elements and metal ions. Notably, this data distinctly cross-validated the results obtained with the M1 sensor indicating the central role of the Golgi apparatus in Mn storage.

CONCLUSIONS AND PERSPECTIVES

We have used a combination of information from confocal imaging studies using a sensitive 'turn-on' fluorescent Mn 2+ sensor and nano-SXRF imaging on cells exposed to sub-cytotoxic levels of Mn to elucidate intra-cellular Mn localization. The Mn 2+ sensor showed fluorescence emission within the Golgi apparatus of HEK293T cells under physiological conditions in the absence of any external Mn exposure. Importantly, the 'turn-on' sensor was able to reveal an increased accumulation of Mn 2+ ions in the Golgi apparatus with increasing levels of Mn 2+ exposure. The confocal microscopy results with the fluorescent sensor indicated a distinct role of the Golgi apparatus as an Mn 2+ storage site under sub-cytotoxic Mn exposure conditions and also hinted toward a possible role of the Golgi apparatus in Mn storage under physiological conditions. The results obtained from confocal microscopy were distinctly confirmed by nano-SXRF, a chemical element imaging technique sensitive to total cellular Mn content. With the experiments on the HEK293T cells in addition to previous studies in HeLa and other neuronal cell lines, the Golgi apparatus can now be established as a compartment that is used for Mn storage and channeling under sub-cytotoxic Mn levels. These experiments have also afforded crucial validation of the Mn 2+ sensing ability of the M1 sensor in living cells. Further, in the future, the capacity of the M1 sensor to detect Mn 2+ in the Golgi apparatus will open possibilities for quantification of Mn levels by using a combination of second generation ratiometric fluorescent sensors with attractive elemental mapping techniques like nano-SXRF. Finally, our

5 Figure 1 .

 51 Figure 1. The confocal image analyses were performed using FIJI (ImageJ, NIH, USA).

Figure 1 .

 1 Figure 1. (A) Left to Right, Confocal images of live HEK293T cells with: no external Mn

Figure 2 .

 2 Figure 2. (A) Representative transmission images of HEK 293T cells after cryo-fixation and

Figure 3 .

 3 Figure 3. SXRF sum-spectra of single cells exposed to 25 µM Mn 2+ (blue curve) and 250 µM Mn 2+ (red curve), after cryofixation and freeze drying. The spectra have been normalized by the

Figure 4 .

 4 Figure 4. (A) Representative transmission images of HEK 293T cells after cryo-fixation and

  Sensor and Nano-SXRF Reveals the Role of the Golgi Apparatus as a Manganese Storage Site

	Sayani Das,

a ‡ Asuncion Carmona, bc ‡ Kaustav Khatua, a ‡ Francesco Porcaro, bc Andrea Somogyi, d Richard Ortega bc and Ankona Datta a

procedures and materials. Unless

	column chromatography purifications were determined by thin layer chromatography (TLC)
	analysis. TLC analyses were performed on silica gel 60 F 254 (Merck & Co., Inc.) and aluminium
	oxide 60 F 254 (Merck & Co., Inc.) TLC plates and the plates were visualized under UV light, 254
	nm and 365 nm. Solvents were evaporated under reduced pressure using a rotary evaporator
	(BÜCHI Labortechnik AG). Low-resolution mass spectral analyses were carried out with an ESI
	probe (positive and negative ion modes) on a liquid chromatography mass spectrometer (LCMS-
	2020, Shimadzu Corp.).
	otherwise mentioned all chemicals were of
	analytical grade, obtained from commercial sources, and used without further purification. Dry
	tetrahydrofuran was obtained by distillation over sodium metal for 1h. Water used for
	experiments was deionized using a Milli Q Integral 3 water purification unit (Millipore Corp.
	Billerica, MA, USA). Silica gel (230-400 mesh size, Merck & Co., Inc.) and Alumina
	(Brockmann grade, S D Fine-Chem Ltd.) were used for column chromatography. Solvents used
	for chromatography were of analytical grade and used without distillation. Eluting systems for

research approach provides a pilot-study on how existing fluorescent sensors can be strategically combined with different validation techniques and protocols to elucidate metal-ion compartmentalization and dynamics.

AUTHOR INFORMATION

Corresponding Author

* Email: ankona@tifr.res.in (AD)

Author Contributions

The manuscript was written through contributions of all authors. All authors have reviewed and given approval to the final version of the manuscript. ‡These authors contributed equally.