Mihai Philippe Besnier

Ionut Andries

Christophe Lemoine

Christophe Lemoine Modele De

Hill Applique

MODELE DE HILL APPLIQUE A LA DETERMINATION DE LA SURFACE EQUIVALENTE D'UN MATERIAU ABSORBANT

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

MODELE DE HILL APPLIQUE A LA DETERMINATION DE LA SURFACE EQUIVALENTE D'UN MATERIAU ABSORBANT

Philippe Besnier, Mihai Ionut Andries, Christophe Lemoine Dans le cadre de mesures en espace libre, l'absorbant est illuminé selon une onde plane dont on ajuste l'angle d'incidence et seule une mesure de réflectivité dans la direction spéculaire est effectuée. Tentons d'imaginer ce qu'une procédure de mesure en chambre réverbérante permettrait d'obtenir. Nous poursuivons l'expérience de pensée qui consiste à considérer que l'absorbant évoqué ci-dessous est quasiidéal. Placé dans une chambre réverbérante, au sein de laquelle on modifie la pondération des modes de propagation au moyen d'un procédé de brassage, l'objet est alors illuminé, en première approximation, selon aucune direction ou polarisation préférentielle. Nous faisons l'hypothèse que nous sommes suffisamment proche d'un champ idéalement désordonné au sens du spectre infini d'ondes planes de Hill [START_REF] Hill | Plane wave integral representation for fields in reverberation chambers[END_REF]. Supposons que l'absorbant soit de forme cubique. De nouveau, nous le considérons comme parfaitement adapté. De plus nous formulons l'hypothèse, certes irréaliste, d'un absorbant dont l'épaisseur de peau tend vers 0. L'absorption s'effectue alors intégralement à la surface de l'absorbant. Dans ces conditions, on montre aisément que sous illumination par le spectre d'ondes planes, un tel absorbant aura pour SEMA : . Dans ces conditions on montre que la SEMA devient [START_REF] Andries | Research of experimental methods to simulate propagation channels in mode-stirred reverberation chamber[END_REF]:

2 43 () 2 objet objet A T a b d               (3)

Dans cette expression

T représente le coefficient moyen de transmission de l'onde incidente dans l'absorbant. La validation de cette estimation a été effectuée au moyen d'un absorbant parallélépipédique émanant de la société SIEPEL dont nous avons obtenu communication des paramètres constitutifs en fonction de la fréquence (permittivité complexe). La surface équivalente d'absorption est évaluée expérimentalement au moyen de l'estimation du contraste de la bande de cohérence obtenue après insertion de l'objet absorbant dans la chambre.

III. PRINCIPE DE MESURE

Le principe de la mesure repose sur une estimation de la bande de cohérence fréquentielle de la chambre. Par définition la bande de cohérence est inversement proportionnelle au coefficient de qualité composite. Tout comme le coefficient de qualité composite, la bande de cohérence peut être considérée comme une variable aléatoire dont on cherche à estimer l'espérance empirique, sans toutefois présupposer de la distribution de cette variable aléatoire ou de celle du coefficient de qualité composite, objet de travaux très récents [START_REF] Arnaut | Probability distribution of the Quality factor of mode-stirred reverberation chamber[END_REF]. Nous nous limitons dans ce qui suit à évaluer l'espérance empirique de la bande de cohérence avec une incertitude limitée afin d'être sensible au contraste associé à la présence d'un absorbant potentiellement de faible volume dans la chambre. Pour ce faire, la bande de cohérence est déterminée au moyen d'échantillons constitués de relevés de mesure en des fréquences consécutives suffisamment rapprochées pour constituer un échantillon corrélé et de suffisamment grande dimension pour estimer la bande de cohérence sur l'intervalle de fréquence correspondant. Cette estimation est réitérée pour un nombre élevé de positions de brasseurs (100 positions). Le coefficient de qualité composite de la chambre et sa relation à la bande de cohérence sont donnés par :

7 , 0 , 3 3 2 cpx c dB B f f f Q    (4)
Cette relation est ici donnée pour une définition spécifique de la bande de cohérence c B symbolisée par l'exposant « cpx, 0,7 » Ceci signifie que la bande de cohérence est définie au sens du seuil égal à 0,7 de la fonction d'autocorrélation de la réponse (complexe) en fréquence de la chambre.

III.1. Evaluation de la bande de cohérence pour une chambre vide

Tout comme en ce qui concerne l'estimation théorique de la SEMA, on admet que la chambre réverbérante est très largement surdimensionnée et que le champ électromagnétique est idéalement désordonné. Dans ces conditions, le coefficient de qualité peut être décrit sous la forme :

T D cP fVE cP fVE Q     2 0 2 0 2 2   (5)
Dans cette expression, f est la fréquence d'analyse, V le volume de la chambre, E 0 l'amplitude moyenne des ondes du spectre d'onde plane au sens de Hill, c la vitesse de la lumière et P D représente la puissance dissipée dans la chambre, identique en l'occurrence à la puissance transmise P T . Le terme représente quant à lui l'impédance d'onde dans le vide. La densité de puissance moyenne S 0 , toujours au sens du spectre d'onde plane est donc implicitement supposée telle que:

 2 0 0 E S  (6)
La puissance émise et dissipée P D dans la chambre peut être définie comme le produit de la densité de puissance moyenne par une surface équivalente d'absorption de la chambre à vide  0 :

0 0  S P D  (7)
Compte tenu de de (4), ( 5) et (6), la puissance dissipée peut être exprimée en fonction de la bande de cohérence de la chambre à vide B c0 :

0 0 3 1   V B cP P c T D  (8)

III.2. Evaluation de la bande de cohérence pour une chambre en présence d'un objet absorbant

Nous émettons d'emblée l'hypothèse que les mécanismes de pertes dans la chambre et dans l'objet sont indépendants. Par conséquent, à puissance transmise P T constante on admet que la puissance dissipée résulte de la combinaison des facteurs de qualité de la chambre vide et de l'objet :

objet vide total Q Q Q 1 1 1   (9)
Dès lors, en présence de l'objet, la répartition de la puissance dissipée s'établit suivant l'équation suivante faisant apparaître la contribution nouvelle de l'objet inséré dans la chambre. Deux antennes ont été simultanément utilisées de sorte à doubler le nombre de réalisations en exploitant les deux ports de l'analyseur de réseau utilisé. Seuls les paramètres S en réflexion sont utilisés pour chaque antenne.

Une configuration typique de mesure est illustrée sur la photo ci-dessous. Deux antennes cornets sont utilisées et émettent principalement dans des directions non en ligne de vue l'une de l'autre, ni de l'objet absorbant sous test. L'absorbant est placé sur un cube de polystyrène qui peut être considéré comme neutre, permettant ainsi à l'objet d'être illuminé sous tous les angles.

Photo 1 Configuration de test typique pour la mesure de SEMA

L'objectif donc dans ce qui suit de comparer l'estimation de la SEMA réalisée à partir des relevés expérimentaux de la réponse fréquentielle de la chambre (11) à l'estimation théorique donnée par (3), dont l'établissement repose sur le modèle du champ idéalement désordonné.

IV. CONFRONTATION THEORIE EXPERIENCE

IV.1. Evaluation de la SEMA d'un absorbant

L'absorbant installé dans la chambre est de type P150 et sa dimension est de 60 60 15  cm 3 . Son épaisseur de peau dans la bande de fréquence 2 GHz à 3 GHz est de l'ordre de 3 cm, selon l'estimation effectuée à partir des paramètres constitutifs de l'absorbant. Les résultats de mesure sont reportés Fig. 1, figure qui fait apparaître en ordonnée la SEMA de cet absorbant dont l'aire est de 1,08 m 2 .

Fig. 1 SEMA de l'absorbant P150 entre 2 et 3 GHz estimée expérimentalement au moyen de la méthode présentée dans cet article (courbe rouge) et de la méthode proposée en [START_REF] Hill | Aperture excitation of electrically large, lossy cavities[END_REF].

Il découle que la SEMA mesurée est environ égale à 0,41 m 2 sur toute la bande. A partir de la permittivité complexe du matériau, on établit que T est environ égal à 0,85 ce qui donne sans le terme correctif entre grandes parenthèses de l'équation (3) une valeur de SEMA environ égale à 0,46. La prise en compte du terme entre crochets permet d'aboutir à la valeur finale de 0,43 donc assez proche de l'estimation provenant de la mesure.

IV.2. Evaluation de la SEMA de plusieurs absorbants accolés

Au-delà, de cette première vérification expérimentale, nous confrontons les évaluations théoriques et expérimentales de la SEMA pour différentes

V. CONCLUSIONS

Cette communication porte sur une évaluation de l'estimation du pouvoir absorbant en chambre réverbérante. L'estimation réalisée à partir de la mesure de la surface équivalente moyenne d'absorption présentée par un objet absorbant de forme simple est extraite expérimentalement à partir de l'hypothèse du spectre d'ondes planes. Ce résultat expérimental est rapproché d'une évaluation entièrement théorique dont les paramètres de calcul initiaux sont les paramètres constitutifs du matériau (permittivité complexe). L'observation des résultats permet de conclure que l'hypothèse du spectre d'ondes planes est raisonnable dans le cadre de l'analyse du pouvoir absorbant d'un objet immergé dans le volume de travail de la chambre. Il convient naturellement de préciser que les bandes de fréquence choisies pour la mesure se situent dans un domaine de fréquence très élevé au regard des dimensions de la chambre. L'estimation de la SEMA rapportée à l'aire d'un absorbant (de forme convexe) représente une réelle opportunité d'évaluation de performances d'absorbants électromagnétiques en chambre réverbérante. Une telle analyse menée à des fréquences pour lesquelles la densité de mode serait trop limitée conduirait inévitablement à des déviations plus importantes par rapport au modèle théorique. Cela rejoint les préoccupations liées à l'obtention d'un champ idéalement diffus en régime de fréquence intermédiaire.

  SEMA relie la puissance absorbée R P par l'objet à la densité de puissance incidente sur l'objet p D . Par contraste, on peut également définir la surface équivalente de diffraction de l'onde. Ainsi, si nous supposons une onde plane en incidence normale à la surface S d'un absorbant pratiquement parfait (adaptation d'impédance et absorption intégrale de l'énergie dans son épaisseur), on trouve S objet   et la surface équivalente de diffraction sera nulle.

A

  représente l'aire de l'absorbant cubique.Ainsi, un absorbant idéal cubique en chambre réverbérante aura pour aire effective la moitié de son aire. Ce résultat peut être étendu immédiatement à un absorbant parallélépipédique.Nous nous intéressons par la suite à la performance réelle d'un absorbant réel, en vue de confrontations expérimentales. Connaissant (à la fréquence de travail) l'épaisseur de peau  , nous supposons l'absorbant de dimension a b d  avec min( , , ) a b d  

III. 3 .

 3 Estimation de la SEMA d'un objet absorbantLa surface équivalente moyenne d'absorption de l'objet peut alors être déduite des estimations de la bande de cohérence en l'absence puis en présence de l'objet. de la bande de cohérence est réalisée au moyen de la mesure du coefficient de réflexion complexe (S 11 ) d'une antenne placée dans la chambre. Pour chaque position de brasseur, le signal reçu par l'antenne d'émission est acquis sur une bande passante de 10 MHz avec une résolution de 50 kHz. Après compensation de la moyenne (assimilable à une erreur d'estimation ou au niveau résiduel d'énergie cohérente), la bande de cohérence correspondante à chaque position de brasseur est déduite de l'analyse de la fonction d'autocorrélation au premier ordre. L'estimation finale de la bande de cohérence est prise comme la moyenne des estimations sur l'ensemble des positions de brasseur.

  combinaisons d'absorbants du même type. Plusieurs absorbants de même taille sont accolés à l'instar de la photo 1 sur laquelle figurent 2 absorbants. L'absorbant P150 est homogène. Par conséquent, du point de vue théorique, la SEMA de l'objet évolue seulement (équation (3)) suivant les nouvelles dimensions a,b,d équivalentes à l'association des absorbants. La figure 2 synthétise les résultats obtenus pour l'association de 2,3 ou 4 absorbants. Pour mémoire, le résultat obtenu avec un seul absorbant est également reporté.

Fig. 2

 2 Fig. 2 Evaluation de la SEMA associée à 1,2,ou 3 ou 4 absorbants. Les courbes oscillantes correspondent aux estimations expérimentales selon (11). Les courbes en traits pointillés correspondent à l'évaluation théorique selon (3)

  Cette communication traite d'une méthode de mesure électromagnétique en chambre réverbérante visant à déterminer l'efficacité d'absorption d'un objet placé à l'intérieur. L'objet est soumis au champ stationnaire créé dans l'enceinte réverbérante entretenu à très haute fréquence vis-à-vis de la dimension de la cavité. Sur le plan théorique, la surface équivalente est évaluée au moyen de l'hypothèse du spectre d'ondes planes de Hill pour un absorbant de forme parallélépipédique dont on connaît les propriétés électromagnétiques. L'absorption d'énergie est évaluée expérimentalement au moyen de l'estimation de la bande de cohérence.

	II. THEORIE
	Nous définissons la surface équivalente moyenne
	d'absorption (SEMA) objet 	d'un objet par :
	nous nous
	attachons à évaluer cette même surface au moyen de la
	mesure de la bande de cohérence moyenne des signaux
	enregistrés dans la chambre sur un ensemble de positions
	de brasseur. Le lien entre la bande de cohérence et la
	puissance moyenne absorbée par l'objet est également
	établi selon l'hypothèse du spectre d'ondes planes.

1 IETR UMR CNRS 6164 -INSA de Rennes, Département Antennes et Dispositifs Hyperfréquences 20 av. des Buttes de Coësmes 35708 Rennes France, E-mail : philippe.besnier@insa-rennes.fr Résumé. I. INTRODUCTION L'énergie électromagnétique moyenne emmagasinée dans une chambre réverbérante dépend de la superposition d'un certain nombre de modes. La présence d'un objet absorbant a pour conséquence la modification du coefficient de qualité moyen associé à l'ensemble des modes excités. Ainsi la détection d'une modification du coefficient de qualité composite peut être à l'origine d'une méthode de détermination du pouvoir absorbant d'un objet placé dans la chambre. Cette proposition a été introduite par M. Andries dans sa thèse soutenue à l'INSA de Rennes en Avril 2013 [1] et synthétisée dans l'article [2]. Nous commençons par définir la notion de surface équivalente moyenne d'absorption et comparons cette notion à la notion habituelle de réflectivité d'un absorbant. Nous montrons en particulier que cette surface est proche à une correction près de la demi-surface visible d'un absorbant parallélépipédique de dimension suffisante que multiplie le coefficient de transmission de l'onde dans le milieu absorbant. Cette évaluation est effectuée au moyen du modèle de Hill. Puis, Différents résultats de mesure contribuent à valider cette estimation. Ils s'appuient sur la mise en oeuvre de matériaux absorbants homogènes dont les paramètres constitutifs sont préalablement connus.


Rebeca Leite

Souza Moreira

Marta K Schütz

Marie Libert

Bernard Tribollet

Vincent Vivier
email: vincent.vivier@upmc.fr

Rebeca Moreira

Influence of hydrogen-oxidizing bacteria on the corrosion of low carbon steel: Local

Keywords: biocorrosion, carbon steel, hydrogen-oxidizing bacteria (HOB), SECM, Local Electrochemical Impedance Spectroscopy

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

Introduction

The safe disposal of radioactive waste is a major concern for the nuclear energy industry. The high level radioactive waste (HLW) should be maintained for years in deep clay formations in order to prevent the migration of radionuclides. Thus, many different kinds of materials such as carbon steel, stainless steel, glass, concrete and clay are employed on the waste repository aiming to act as a multi-barrier system [START_REF] David | Analogue archéologique et corrosion, Collection Sciences et Techniques[END_REF]. However, the anoxic corrosion of the metallic materials is expected due to the changes on the environmental conditions around the buried structures such as resaturation of the repository with time. In this context, corrosion products like iron oxides (i.e. magnetite, Fe 3 O 4 ) or hydroxides, and dihydrogen (H 2 ) are also expected to be formed according to the following reactions (Eqs. 1-8) [START_REF] David | Analogue archéologique et corrosion, Collection Sciences et Techniques[END_REF]:

In aerobic condition

The production of dihydrogen poses two major problems. On the one hand, it represents a significant threat to the repository when accumulated for a long time in the surrounding clay because it may damage the barrier properties of the geological formation, affecting the safety of the repository. On the other hand, dihydrogen also represents a new energy source for microbial growth, especially in such anoxic environments with low content of biodegradable organic matter.

Several studies have already pointed out the possibility of microbiological life in the deep geological disposal [START_REF] Stroes-Gascoyne | Microbial occurrence in bentonite-based buffer, backfill and sealing materials from large-scale experiments at AECL's Underground Research Laboratory[END_REF][START_REF] Pedersen | Investigations of subterranean bacteria in deep crystalline bedrock and their importance for the disposal of nuclear waste[END_REF][START_REF] Stroes-Gascoyne | Microbial analysis of samples from the tunnel sealing experiment at AECL's Underground Research Laboratory[END_REF][START_REF] Urios | Microbial diversity of the 180 million-yearold Toarcian argillite from Tournemire, France[END_REF], and both biological activity and biofilm formation may influence the metallic corrosion rate. These phenomena are usually known as microbiologically influenced corrosion (MIC), which can represent a huge problem when promoting local metal dissolution by different mechanisms which vary with the microbial species and chemistry of the colonized metal surface [START_REF] Beech | Biocorrosion: towards understanding interactions between biofilms and metals[END_REF]. For instance, in such confined environment, some iron-reducing bacteria (IRB) are able to use simultaneously dihydrogen as electron donor and ferric iron as electron acceptor for their anaerobic respiration [START_REF] Lovley | Dissimilatory Fe(III) and Mn(IV) reduction[END_REF][START_REF] Libert | Molecular hydrogen: An abundant energy source for bacterial activity in nuclear waste repositories[END_REF]. In this study, Shewanella oneidensis was chosen as an IRB and hydrogen-oxidizing bacteria HOB model microorganism. It is a facultative anaerobic bacterium [START_REF] Perez-Gonzalez | Magnetite biomineralization induced by Shewanella oneidensis[END_REF] able to use different compounds as electron donor, such as lactate, formate, pyruvate, amino acids and also dihydrogen [START_REF] Nealson | Breathing metals as a way of life: geobiology in action, Antonie Van Leeuwenhoek[END_REF]. According to El-Naggar et al. [START_REF] El-Naggar | Electrical transport along bacterial nanowires from Shewanella oneidensis MR-1[END_REF] the respiration rate of Shewanella oneidensis strain MR-1 is 2.6x10 6 electrons per cell per second using lactate as electron donor. In addition, some mechanisms of IRB species using electron transfers from solid substrates have already been described in the literature, such as:  the use of mediators which can act as electron shuttles transferring electrons from the cells to the acceptors compounds [START_REF] Brutinel | Shuttling happens: soluble flavin mediators of extracellular electron transfer in Shewanella[END_REF][START_REF] Lovley | Microbial fuel cells: novel microbial physiologies and engineering approaches[END_REF];  the direct contact of cells with solid substrate through multihemes cytochromes at the external membrane [START_REF] Du | A state of the art review on microbial fuel cells: A promising technology for wastewater treatment and bioenergy[END_REF];  the use of conductive intracellular filaments (i.e. nanowires) [START_REF] El-Naggar | Electrical transport along bacterial nanowires from Shewanella oneidensis MR-1[END_REF]. It is well-known that the presence of microorganisms can influence the corrosion rate [START_REF] Beech | Biocorrosion: towards understanding interactions between biofilms and metals[END_REF][START_REF] Jones | A thermodynamic interpretation of microbiologically influenced corrosion[END_REF][START_REF] Busalmen | New evidences on the catalase mechanism of microbial corrosion[END_REF][START_REF] Schrott | Electrochemical insight into the mechanism of electron transport in biofilms of Geobacter sulfurreducens[END_REF][START_REF] Silva | Electron transfer between hydrogenase and 316L stainless steel: identification of a hydrogenase-catalyzed cathodic reaction in anaerobic mic[END_REF][START_REF] Beech | Accelerated low water corrosion of carbon steel in the presence of a biofilm harbouring sulphate-reducing and sulphur-oxidising bacteria recovered from a marine sediment[END_REF], but is not well elucidated if IRB are able to induce local corrosion. The overall influence of microorganism on corrosion mechanism is still under debate since it depends on many factors [START_REF] Beech | Biocorrosion: towards understanding interactions between biofilms and metals[END_REF].

Electrochemical techniques such as cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS) were already proved to be powerful tools for corrosion investigations. Valuable parameters can be gathered about the kinetics of interfacial processes from the measurements of current or potential. However, these results are obtained as an average behavior of the whole interface, which render difficult the elucidation of the local corrosion mechanisms [START_REF] Isaacs | The localized breakdown and repair of passive surfaces during pitting[END_REF][START_REF] Lillard | A novel method for generating quantitative local electrochemical impedance spectroscopy[END_REF][START_REF] Galicia | Corrosion study of an AZ91 magnesium alloy by EIS and LEIS[END_REF][START_REF] De Assis | Assessment of electrochemical methods used on corrosion of superduplex stainless steel[END_REF]. Therefore, the use of local techniques is needed to describe the interface reactivity.

The objective of this study is to characterize the electrochemical interface by both local and global techniques during biocorrosion of low carbon steel in presence of Shewanella oneidensis used as model of IRB and HOB. Scanning electrochemical microscopy (SECM), which has been widely described by and subject of numerous reviews [START_REF] Mirkin | Scanning electrochemical microscopy in the 21st century. Update 1: five years after[END_REF][START_REF] Keddam | Progress in Scanning Electrochemical Microscopy by Coupling with Electrochemical Impedance and Quartz Crystal Microbalance[END_REF][START_REF] Wittstock | Scanning electrochemical microscopy for direct imaging of reaction rates[END_REF], and local electrochemical impedance spectroscopy pioneered by Isaacs et al. [START_REF] Lillard | A novel method for generating quantitative local electrochemical impedance spectroscopy[END_REF][START_REF] Zou | A high-resolution probe for localized electrochemical impedance spectroscopy measurements[END_REF] were used as electrochemical techniques. Carbon steel has been considered as candidate material for the multi-barrier system due to the low corrosion rate under reducing conditions. In a previous study [START_REF] Esnault | Metallic corrosion processes reactivation sustained by iron-reducing bacteria: Implication on long-term stability of protective layers[END_REF], corrosion products, iron oxides (e.g. magnetite) and H 2 were shown to favor the hydrogen-oxidizing IRB development in the context of active corrosion of metallic radioactive waste containers. Moreover, the size of bacterial cells is in the range of few micrometers to few tens micrometers, which makes possible to perform local electrochemical measurements even in the biofilm environment. A microelectrode can also be used for generating locally H 2 in the close vicinity of the bacterial cells.

Experimental

Bacterial culture

The cultures of Shewanella oneidensis strain MR-1 (ATCC 700550 TM ) were obtained aerobically at the beginning of the stationary growth phase in Luria Bertani Broth (LB) medium (5 g.L -1 NaCl, 10 g.L -1 tryptone, 5 g.L -1 yeast extract) after 24 h at 30°C. Shewanella oneidensis cells were harvested from the LB medium by centrifugation (4000 rpm for 20 min), washed once with sterile minimal medium (M1) and then inoculated in electrochemical reactor (initial concentration of 10 8 cells.mL -1 counted by THOMA counting chamber). The chemically defined minimal medium (M1) was prepared according to Esnault et al. [START_REF] Esnault | Metallic corrosion processes reactivation sustained by iron-reducing bacteria: Implication on long-term stability of protective layers[END_REF]. The composition consisted in a mixture of 9 mM (NH 4 ) 2 SO 4 , 2 mM NaHCO serine), and vitamins (0.08 mM nicotinic acid, 0.01 mM thiamine-HCl, 0.40 µM biotine). The pH was adjusted to ca. 7 with NaOH and then the medium was sterilized by autoclaving (120°C for 20 min), except for the thermolabile components (e.g. amino acids) which were filter-sterilized (0.22 µm). For local impedances experiments this medium was prepared without phosphate ions in its composition.

Carbon steel coupons

Low carbon steel coupons A37 (0.12% C, 0.22% Si, 0.62% Mn, 0.008% Al, 0.012% S and P, 0.02% Ni, 0.03% Cr, 0.04% Cu, 0.005% Co and <0.005% Ti) were obtained from the CEA (Commissariat à l'Energie Atomique et aux Energies Alternatives -France). Rod-shaped coupons (1 cm in diameter) were laterally insulated with a cathaphoretic paint and then molded in a diallylphthalate glass-fiber resin (Presi), leaving a disk area of 0.79 cm 2 exposed to the solution. Prior to each experiment, the electrode was polished with emery paper (P600 grit SiC) and sterilized with ethanol by sonication for 15 min.

Electrochemical experiments 2.3.1. Scanning electrochemical microscopy (SECM)

Preliminary experiments for the detection of H 2 were performed in a 0.5 M sulfuric acid solution with a platinum electrode of 0.5 cm in diameter as substrate and a platinum microelectrode of 20 µm in radius.

For the experiments related to the study of carbon steel corrosion, the electrochemical measurements were performed with a home-made scanning electrochemical microscope (SECM), which consisted in a 4-electrode cell configuration depicted in Scheme 1, elaborated specifically for anaerobic and sterile conditions. A double-wall glass cell thermostated at 30°C allowed the carbon steel working electrode (WE1) to be placed in the bottom of the cell in a face up position. The reference and the counter electrodes consisted in a saturated calomel reference electrode (SCE) and a platinum gauze, respectively. The cell was closed with a Teflon cover where a flexible nitrile bracket was adapted. Throughout this flexible support, a platinum tip of 50 µm in diameter, acting as a second mobile working electrode for generating H 2 in solution (see Results and discussion section), was placed perpendicular to WE1. A home-made bi-potentiostatic workstation coupled with high gain and low noise current to voltage converters were used. The microelectrode was positioned with the help of motorized stages allowing a spatial resolution of 200 nm along the 3-axis and was driven by a motion controller (Newport). All experiments were computer controlled with a Labview software [START_REF] Gabrielli | Electrochemical Impedance Spectroscopy Investigations of a Microelectrode Behavior in a Thin-Layer Cell: Experimental and Theoretical Studies[END_REF][START_REF] Gabrielli | Scanning Electrochemical Microscopy Imaging by Means of High-Frequency Impedance Measurements in Feedback Mode[END_REF]. The disassembled cell and the electrodes were sterilized prior to experiments with 70% ethanol solution for 20 min; and then rinsed with sterilized water in laminar flow chamber followed by UV irradiation for 15 min. Whenever possible, the instrumentation was sterilized in an autoclave. The cell was assembled under sterile conditions and 260 mL of M1 medium was introduced and deaerated for 2 h under N 2 atmosphere. The N 2 flow was maintained at the headspace of the cell (near to the liquid surface) so as to ensure the anaerobic conditions during the whole duration of the electrochemical measurements.

The contact point between the substrate and the probe (i.e. the distance 0) was determined by performing an approach curve [START_REF] Bard | Scanning electrochemical microscopy. Introduction and principles[END_REF][START_REF] Kwak | Scanning electrochemicam microscopy. Theory of the feedback mode[END_REF]. Then, the SECM was used in generation / collection mode [START_REF] Wittstock | Imaging localized reactivities of surfaces by scanning electrochemical microscopy[END_REF][START_REF] Sanchez-Sanchez | Scanning Electrochemical Microscopy. 60. Quantitative Calibration of the SECM Substrate Generation/Tip Collection Mode and Its Use for the Study of the Oxygen Reduction Mechanism[END_REF] for investigating the role of H 2 for different distances between the probe and substrate.

Here Scheme 1

Local electrochemical impedance spectroscopy (LEIS)

Similarly to the SECM, LEIS consisted in using a micro probe for measuring the local current density [START_REF] Lillard | A novel method for generating quantitative local electrochemical impedance spectroscopy[END_REF][START_REF] Frateur | Experimental Issues Associated with Measurement of Local Electrochemical Impedance[END_REF][START_REF] Huang | The apparent constant-phase-element behavior of an ideally polarized blocking electrode a global and local impedance analysis[END_REF][START_REF] Huang | Local electrochemical impedance spectroscopy: A review and some recent developments[END_REF]. The two devices require some similar apparatus, in particular for positioning the probe in the vicinity of the substrate. The LEIS is based on the measurement of the local potential difference in solution with a bi microreference electrode (dual Ag/AgCl microelectrodes) positioned close to the substrate as sketched in Scheme 2. These microreference electrodes were obtained from the anodization of silver microelectrodes in a potassium chloride solution. The local AC-current density ( ) loc i  was obtained through the Ohm's law using [START_REF] Isaacs | The effect of height on the current distribution measured with a vibrating electrode probe[END_REF]:

( ) ( ) probe loc V i d      (9)
where  is the electrolyte conductivity,

( ) probe V  
is the AC potential difference between the two probes, and d is the distance between the two probes. The local impedance (z) involves the electrode potential measured with respect to a reference electrode located far from the electrode surface and the local current according to [START_REF] Huang | The apparent constant-phase-element behavior of an ideally polarized blocking electrode a global and local impedance analysis[END_REF][START_REF] Huang | Local electrochemical impedance spectroscopy: A review and some recent developments[END_REF][START_REF] Isaacs | The effect of height on the current distribution measured with a vibrating electrode probe[END_REF]:

ref ( ) ( ) ( ) ( ) ( ) loc probe V V d z i V            (10)
where re f ) ( ~   V
represents the AC potential difference between the electrode surface and the reference electrode in the bulk solution. Similarly to other scanning probe techniques, the spatial resolution of the LEIS is governed by the size of the probe and the distance between the probe and the steel substrate. The measurements were carried out with a four-channel frequency response analyzer (Solartron 1254) and the potential difference between the two microelectrodes was amplified with a home-made differential amplifier. For this study, a specific tri-microelectrode was devised (Scheme 2) allowing the generation of H 2 and local microbiological activity to be induced with the platinum microelectrode (similarly to the generation / collection mode of the SECM), and the simultaneous local electrochemical impedance measurement with the two Ag/AgCl microreference electrodes.

Here Scheme 2

Characterization of the coupon surface

Steel coupon surfaces were analyzed at the end of the experiments by scanning electron microscopy (SEM). These investigations were performed with a Leica Stereoscan 440 microscope coupled with EDS elemental semi-quantitative analyses (Princeton Gamma-Tech) at 20 keV and 5.3 nA. The samples were slightly rinsed with deionised water, dried with N 2 gas and stored in plastic containers until analysis. The coupons were coated with carbon prior to SEM analysis.

Results and discussions

The results of the preliminary experiments related to the generation / collection of H 2 in solution are illustrated in the Figure 1. These experiments were performed in a 0.5 M sulfuric acid solution with a 0.5 cm in diameter platinum electrode as substrate. The potential of the substrate was swept at 15 mVs -1 in the H 2 evolution reaction domain (Fig. 1a), whereas the dissolved H 2 generated by the substrate was simultaneously monitored as a function of time with a platinum microelectrode biased at 0.3 V (vs. SCE) for different tip-to-substrate distance (Figure 1b). The current peak observed after about 11 seconds corresponds to the beginning of the reverse scan of the Pt substrate. It is noteworthy that the current of the substrate does not depend on the tip-to-substrate distance (Figure 1a). Conversely, the response of the probe strongly depends on this distance. First, there is a further delay in the response of the probe, which corresponds to a longer time for the electrogenerated species to reach the microelectrode when it is positioned far from the substrate. Second, due to the concentration gradient of these species from the Pt substrate to the bulk solution, the peak intensity of the current recorded at the microelectrode decreases with the distance. However, these experiments clearly evidence the possibility of dosing H 2 dissolved in solution with a microelectrode positioned at a distance smaller than few microelectrode radii.

Here Fig. 1 Figure 2 shows the CV curves of a platinum probe at 15 mVs -1 positioned at 80 µm of the interface in abiotic (blue curve) and biotic (red curve) conditions for 72 h and 24 h after inoculation, respectively. These two curves were obtained successively during the same experiment and at the same location for the probe. During the cathodic scan, H 2 is generated at the probe and diffuses towards the interface where it can be consumed by the bacteria. The anodic scan corresponds to the re-oxidation of H 2 remaining in solution. It is noteworthy that the electrochemical cell formed by the microelectrode and the substrate is a thin layer in that case. This is the reason why the anodic peaks observed for both cases are ascribed to the dihydrogen oxidation. Moreover, the amount of H 2 determined from the peak area is larger in abiotic conditions, indicating a consumption of H 2 by the bacteria.

Here Fig. 2 This result is confirmed by Figure 3 on which the exchanged charge for different microelectrode-to-substrate distances is reported for biotic (red curve) and abiotic (blue curve) conditions. Interestingly, the two curves exhibit very similar behavior and the difference between the two curves provides an evaluation of the H 2 consumption by the bacteria. A mean value of about 0.12 µC was obtained for the amount of H 2 consumed. From the Faraday's law, this corresponds to about 6 10 -13 mol of H 2 , and taking into account the duration of the CV experiment for both generation and dosing of H 2 , this correspond to a H 2 consumption rate of about 10 -14 mols -1 for biotic conditions. For comparison, the charge exchanged during the H 2 production by the tip was about 9 µC showing that only 1 to 1.5% of the H 2 generated is involved in the bacterial activity.

Here Fig. 3 Figure 4 shows the global impedance diagrams of the steel coupon for biotic conditions. These experiments were performed with H 2 generation by the probe polarized at -1 V / ECS and generating a dc current of about 50 nA, as shown in the experimental setup of the Scheme 2. The high frequency loop between 65 kHz and 10 kHz is ascribed to artefacts as already described in the literature [START_REF] Tran | Artefacts in electrochemical impedance measurement in electrolytic solutions due to the reference electrode[END_REF], and the electrochemical system can be readily described with two time constants. The high frequency time constant (10 kHz < f < 100 Hz) is characterized by an amplitude of about 30 cm 2 and does not significantly varies with time. Such value is small and cannot be attributed to a charge transfer resistance, but to a thin corrosion layer products which have already been described for carbon steel in sterile artificial sea water [START_REF] Castaneda | SRB-biofilm influence in active corrosion sites formed at the steel-electrolyte interface when exposed to artificial seawater conditions[END_REF]. In addition, the analysis of this flattened capacitive loop allows a constant phase element to be determined, and from the used of Brug formula [START_REF] Brug | The analysis of electrode impedances complicated by the presence of a constant phase element[END_REF][START_REF] Hirschorn | Determination of effective capacitance and film thickness from constant-phase-element parameters[END_REF], a capacitance is obtained (Figure 5). This capacitance is about 7 µFcm -2 and is time independent. Assuming a permittivity of 10 for the corrosion products, the thickness of the layer is in the range of 1 to 2 nm. As shown in Figure 4, in the low frequency domain (f < 100 Hz), a second time constant can also be evidenced. The amplitude of the loop is independent of the duration of the experiment (3000 cm 2 ) and is ascribed to the charge transfer resistance of the steel dissolution process. The fitting of this low frequency time constant also needs the use of a constant phase element (CPE), and as previously mentioned, the use of Brug formula allows the double layer capacitance to be obtained. Its variations are reported in Figure 6 and show an increase with time after 60 hours (whereas in abiotic condition, a continuous increase with time was obtained) from 300 µFcm -2 to 750 µFcm -2 after five days. Such unusual high values for double layer capacitance can be explained by the accumulation of corrosion product over the electrode surface and the 3D architecture of the interface in presence of biological material [START_REF] Castaneda | SRB-biofilm influence in active corrosion sites formed at the steel-electrolyte interface when exposed to artificial seawater conditions[END_REF][START_REF] Nielsen | Microbial corrosion of carbon steel by sulfate-reducing bacteria: electrochemical and mechanistic approach[END_REF]. Thus, the constant value of the charge transfer resistance indicates that the carbon steel is dissolving at a constant rate, whereas the increase of the double layer capacitance is linked to the growth of the system formed by the bacteria and the corrosion products.

Here Fig. 4 Here Fig. 5 Here Fig. 6 During the same set of experiments, local impedance measurements were performed on various locations above the steel substrate. Figure 7 shows some examples of LEIS diagrams obtained at the electrode center (Figure 7a) and at 1 mm from the center (Figure 7b). Both set of diagrams are very similar. In the high frequency domain, the inductive behavior is due to the cell geometry, as already described by Frateur et al. [START_REF] Frateur | Experimental Issues Associated with Measurement of Local Electrochemical Impedance[END_REF][START_REF] Frateur | Local electrochemical impedance spectroscopy: Considerations about the cell geometry[END_REF]. This inductive component hides the first capacitive loop, and only the second loop corresponding to the charge transfer can be used for the fitting. Interestingly, the values of the charge transfer resistance are independent of both time and location. This indicates that from a local point of view (the spatial resolution of the setup used is about 100 µm) the reactivity of the interface does not depend on the position. In addition, the double layer capacitance obtained from local measurements shows the same trends with time (i.e. a continuous increase), but this value remains constant for various location at different times. This is in agreement with the charge transfer resistance variation and suggests that Shewanella oneidensis influences the overall reaction rate in this environment, but no local corrosion could have been evidenced from the analysis of electrochemical results. In addition, this was supported by post-mortem SEM observations as shown in Figure 8. The visual appearance of steel is characteristic of a generalized corrosion and a close examination of the surface after different experiments never allowed evidencing localized corrosion.

Here Fig. 7 Here Fig. 8

Conclusions

This work shows that local electrochemical techniques are applicable for the investigation of biocorrosion using Shewanella oneidensis as model of iron-reducing bacteria as well as hydrogen-oxidizing bacteria. The dissolved H 2 concentration in H 2 SO 4 solution can be measured or controlled by SECM with the generation / collection mode when the distance between the tip and the substrate is smaller than few microelectrode radii. The charge of the H 2 re-oxidation, in the thin layer between the probe and the substrate, was compared for biotic and abiotic conditions. A small charge was observed for biotic conditions indicating that an amount of the H 2 generated by the probe was consumed by Shewanella oneidensis. Therefore, the use of the scanning electrochemical microscopy allowed evidencing the H 2 consumption by the bacteria. Moreover, a H 2 consumption rate of about 10 -14 mol s -1 was measured when only corrosion products were used as energetic substrates.

Two capacitive loops were observed in the global impedance measurements from the experiments performed with the M1 medium for biotic conditions. The high frequency loop can be attributed to a thin layer of corrosion products due to the small and constant values of capacitance (9.5 µFcm -2 ). A thickness in the range of 1 to 2 nm was estimated for this layer. The charge transfer resistance of the steel dissolution process was attributed to the low frequency range. The high capacitance values measured are caused by the spatial distribution of species in the biofilm in combination with growth of a film of corrosion products. General corrosion was clearly evidenced by local electrochemical results and SEM observations. Thus, starting from the general idea that biocorrosion is associated to localized corrosion; this work clearly shows that in the case of Shewanella oneidensis, generalized corrosion predominates for short-time experiments.

These local descriptions of electrochemical processes offer new perspectives for investigating the relevant activity of a given microorganism, or to studying the concomitant effect of actual environment in combination to the presence of bacteria.

Acknowledgements

This research was funded by the European Community's Seventh Framework Programme (FP7/2007-2013) by the BIOCOR project. The authors wish to thank Damien Féron (CEA Saclay, France) for carbon steel samples and Françoise Pillier (LISE, UPR15, France) for SEM analysis.

• before inoculation; • after 4 h; ▲ after 44 h; ▼ after 68 h; ► after 93 h; ◄ after 116 h

• before inoculation; • after 4 h; ▲ after 44 h; ▼ after 68 h; ► after 93 h; ◄ after 116 h

Figure captions Scheme 1 :Scheme 2 :

 12 Figure captions

Figure 1 :

 1 Figure 1: Dihydrogen detection with the SECM in H 2 SO 4 solution. The potential of the Pt substrate was swept in the hydrogen reduction reaction domain at 15 mVs -1 (curve a), whereas the platinum probe was biased at 0.3 V (vs. SCE) for dissolved dihydrogen detection (curve b), with the tip-to-substrate distance as a parameter. The minimum in current observed after 11 seconds on curves of Figure (a) corresponds to the inversion of the potential scan direction.

Figure 2 :

 2 Figure 2: Cyclic voltammograms of a Pt microelectrode of 50 µm in diameter at 15 mVs -1 positioned at 80 µm from the steel substrate in abiotic (curve a) and biotic (curve b) conditions for 72 h and 24 h after inoculation, respectively. The curves presented are a zoom of the anodic domain.

Figure 3 :

 3 Figure 3: Evolution of the amount of H 2 oxidized (given as charge) at the Pt microelectrode during the backward scan of potentials as a function of the tip-to-substrate distance. The red curve was measured in abiotic conditions and the blue one in biotic conditions. Symbols are experimental measurements.

Figure 4 :

 4 Figure 4: Evolution of the global electrochemical impedance diagrams at the corrosion potential in presence of bacteria with time as parameter. Electrode surface A = 0.79 cm 2 .

Figure 5 :

 5 Figure 5: Evolution of the capacitance in high frequency range.

Figure 6 :

 6 Figure 6: Evolution of the capacitance in low frequency range.

Figure 7 :Figure 8 :

 78 Figure 7: Evolution of the local electrochemical impedance diagrams in presence of bacteria with time as parameter. (a) at the electrode centre, and (b) at 1 mm from the electrode centre.Electrode surface A = 0.79 cm2 . ○ before inoculation; ▲ after 1 h; ▼after 4 h; ◊ after 44 h; ◄ after 68h; ► after 93 h; after 116 hFigure 8: SEM image of the carbon steel substrate after 5 days of immersion in the M1 solution in biotic conditions.

Figure 1 :

 1 Figure 1: Dihydrogen detection with the SECM in H 2 SO 4 solution. The potential of the Pt substrate was swept in the hydrogen reduction reaction domain at 15 mVs -1 (curve a), whereas the platinum probe was biased at 0.3 V (vs. SCE) for dissolved dihydrogen detection (curve b), with the tip-to-substrate distance, d, as a parameter. The minimum in current observed at 11 seconds on curves of Figure (a) corresponds to the inversion of the potential scan direction. 1-d=5µm; 2-d=10µm; 3-d=25µm; 4-d=50µm; 5-d=100µm; 6-d=250µm.

Figure 2 :

 2 Figure 2: Cyclic voltammograms of a Pt microelectrode of 50 µm in diameter at 15 mVs -1 positioned at 80 µm from the steel substrate in abiotic (curve a) and biotic (curve b) conditions for 72 h and 24 h after inoculation, respectively. The curves presented are a zoom of the anodic domain.

Figure 3 :

 3 Figure 3: Evolution of the amount of H 2 oxidized (given as charge) at the Pt microelectrode during the backward scan of potentials as a function of the tip-to-substrate distance. The red curve was measured in abiotic conditions and the blue one in biotic conditions. Symbols are experimental measurements.

Figure 4 :

 4 Figure 4: Evolution of the global electrochemical impedance diagrams at the corrosion potential in presence of bacteria with time as parameter. Electrode surface A = 0.79 cm 2 .

Figure 5 :

 5 Figure 5: Evolution of the capacitance in high frequency range.

Figure 6 :

 6 Figure 6: Evolution of the capacitance in low frequency range.

Figure 7 :

 7 Figure 7: Evolution of the local electrochemical impedance diagrams in presence of bacteria with time as parameter. (a) at the electrode centre, and (b) at 1 mm from the electrode centre.Electrode surface A = 0.79 cm 2 . ○ before inoculation; ▲ after 1 h; ▼after 4 h; ◊ after 44 h; ◄ after 68h; ► after 93 h; after 116h

Figure 8 :

 8 Figure 8: SEM image of the carbon steel substrate after 5 days of immersion in M1 solution in biotic conditions.

  3 , 0.8 mM MgSO 4 .7H 2 O, 0.4 mM CaCl 2 .2H 2 O, 45 µM H 3 BO 3 , 10 µM NaCl, 4 µM FeSO 4 .7H 2 O, 5 µM CoSO 4 .7H 2 O, 5 µM NiSO 4 .6H 2 O, 3 µM Na 2 MoO 4 .2H 2 O, 11 µM Na 2 SeO 4 , 1 µM MnSO 4 .H 2 O, 0.8 µM ZnSO 4 .7H 2 O, 0.2 µM CuSO 4 .5H 2 O, 17 mM HEPES buffer, amino acids (0.11 mM arginine, 0.13 mM glutamate, 0.19 mM

Author CVs

Rebeca Moreira is graduated in Food Engineering with a Master degree in Quality Control in Food and Drink Industries in Rio de Janeiro, Brazil. Started the PhD study in 2010 as a member of the BIOCOR Program at the Laboratory of Interfaces and Systems Electrochemistry (University Pierre et Marie Curie), Paris, France. The subject of her work is the study of the biocorrosion of carbon steel using local electrochemical techniques.

Marta Kerber Schütz received a BS degree in Industrial Chemistry from Catholic University of Rio Grande do Sul, Porto Alegre, Brazil in 2007; completed her MSc in Materials Science and Engineering in March 2010 at the same University. Now she is a PhD student at the French Commission for Atomic Energy and Alternative Energies (CEA). She is working on biocorrosion phenomena implicated on the radioactive waste geological disposal. The overall objective of her study is to better understand the impact of bacterial activities on the corrosion products (specially hydrogen and iron (hydr)oxides) and on the rate of anoxic corrosion.

Marie


Antoine G Godin

Brahim Lounis

Laurent Cognet
email: laurent.cognet@u-bordeaux.fr

Super-resolution microscopy approaches for live cell imaging

Keywords: super-resolution microscopy, nanoscopy, fluorescence microscopy, live-cell imaging, single molecule detection, localization microscopy Live cell super-resolution imaging

By delivering optical images with spatial resolutions below the diffraction limit several superresolution fluorescence microscopy techniques opened new opportunities to study biological structures with details approaching molecular structure sizes. They have now become methods of choice for imaging proteins and their nano-scale dynamic organizations in live cells. In this minireview, we describe and compare the main far-field super-resolution approaches that allow studying endogenous or overexpressed proteins in live cells.

Introduction

The decryption of cell functions and sub-cellular processes has constantly benefited from advances in microscopy. In particular, the developments of fluorescence microscopy and of numerous fluorescent probes allowing the study of specific biomolecules at work in their native environment were instrumental to the advance of live cell mechanisms investigations. The optical resolution of microscopes is limited by the diffraction of light which commonly sets a limit of ~λ/2 in far-field microscopy. By delivering optical images with spatial resolutions below the diffraction limit, super-resolution fluorescence microscopy offered new promises to study molecular processes with greater details than with conventional microscopies [START_REF] Hell | Far-field optical nanoscopy[END_REF][START_REF] Huang | Super-resolution fluorescence microscopy[END_REF]. Most of these methods rely on the control of the number of emitting molecules in specific imaging volumes. This can be achieved by controlling local emitter fluorescent state populations or the labeling densities of fluorescing probes at any given time during the image acquisition process. In this mini-review, we will discuss the key features of super-resolution techniques used for live cell studies. We schematically divide them into three major groups: those based on highly localized fluorescence emission volumes, based on structured illumination and based on single-molecule localizations. A didactic representation of the three families of super-resolution approaches is presented in Figure 1.

Super-resolution based on highly localized fluorescence emission volumes.

In a far-field confocal microscope, the effective fluorescence volume can be reduced below the diffraction limit(3) by using saturable optical processes which de-excite emitters formerly excited by a focused laser beam. These processes consist in preventing fluorescence emission from specific regions of the excitation beam by driving the molecules in these regions between bright and dark states using a depletion light beam. An elegant and efficient strategy consists in using stimulated emission by a high intensity (>MW/cm 2 ) doughnut-shaped laser beam superimposed with the focused excitation laser beam. Doing so, fluorescence emission from emitters in peripheral regions of the excitation beam is completely prevented. This process was coined STimulated Emission Depletion (STED) [START_REF] Hell | Breaking the diffraction resolution limit by stimulated emission: stimulated-emission-depletion fluorescence microscopy[END_REF]. A doughnut-shape depletion beam is the simplest design but in general, any depletion beam featuring a spatial intensity distribution with one or several intensity zeroes can be used to perform STED images.

To generate a super-resolved image with STED which is based on local excitation volumes, it is thus necessary to scan the excitation/depletion effective volumes over the sample in a deterministic point by point or parallelized scanning schemes [START_REF] Bingen | Parallelized STED fluorescence nanoscopy[END_REF][START_REF] Yang | Large parallelization of STED nanoscopy using optical lattices[END_REF]. STED was successfully applied in several live samples to study slow morphing and movements of organelles like reticulum endoplasmic or microtubules [START_REF] Hein | Stimulated emission depletion (STED) nanoscopy of a fluorescent protein-labeled organelle inside a living cell[END_REF], sub-cellular organization in live cells [START_REF] Westphal | Video-rate far-field optical nanoscopy dissects synaptic vesicle movement[END_REF], synaptic structures in live samples [START_REF] Westphal | Video-rate far-field optical nanoscopy dissects synaptic vesicle movement[END_REF][START_REF] Nagerl | Live-cell imaging of dendritic spines by STED microscopy[END_REF][START_REF] Tonnesen | Spine neck plasticity regulates compartmentalization of synapses[END_REF]. For live cell studies, one should bear in mind that relatively high laser powers are needed in STED, especially when using continuous wave laser beams (e.g. ~MW/cm 2 (10)). Using pulsed excitation beam together with time-gating detection allowed a ~2-3 fold reduction in laser power [START_REF] Vicidomini | Sharper low-power STED nanoscopy by time gating[END_REF]. In addition, photobleaching is a limiting factor for longterm live sample imaging because each fluorescent molecule undergoes a large number of exciting/de-exciting cycles in the depletion beam.

An approach similar to STED using much lower intensities to deplete emitting molecular levels (~kW/cm 2 )( 12) is based on reversible photoswitching of marker proteins between a fluorescence-activated and a non-activated state [START_REF] Hofmann | Breaking the diffraction barrier in fluorescence microscopy at low light intensities by using reversibly photoswitchable proteins[END_REF][START_REF] Dedecker | Subdiffraction imaging through the selective donut-mode depletion of thermally stable photoswitchable fluorophores: numerical analysis and application to the fluorescent protein Dronpa[END_REF][START_REF] Grotjohann | Diffraction-unlimited all-optical imaging and writing with a photochromic GFP[END_REF], whereby one of the transitions is accomplished by means of a spatial intensity distribution featuring a zero. This generalized approach was named after the acronym REversible Saturable OpticaL Fluorescence Transitions (RESOLFT). The development of bright photostable switchable fluorophores and fluorescent proteins were particularly instrumental in the development of these techniques [START_REF] Dedecker | Subdiffraction imaging through the selective donut-mode depletion of thermally stable photoswitchable fluorophores: numerical analysis and application to the fluorescent protein Dronpa[END_REF][START_REF] Grotjohann | Diffraction-unlimited all-optical imaging and writing with a photochromic GFP[END_REF][START_REF] Patterson | A photoactivatable GFP for selective photolabeling of proteins and cells[END_REF].

Importantly, fluorescent proteins provide specific 1:1 protein labeling and offer the possibility of intracellular live-cell imaging.

Structured illumination microscopy (SIM).

Structured Illumination Microscopy (SIM) is based on standard wide-field microscopy and is compatible with most standard fluorophores and labeling protocols. It uses non-uniform illuminations with known spatial patterns (e.g. originally a sinusoidal grid, but other illumination distributions can also be used [START_REF] Gao | Noninvasive imaging beyond the diffraction limit of 3D dynamics in thickly fluorescent specimens[END_REF]). From multiple acquisitions (e.g. 9 images: 3 phase shifts for 3 pattern orientations [START_REF] Kner | Superresolution video microscopy of live cells by structured illumination[END_REF]), high spatial frequency information is retrieved with dedicated algorithm, otherwise inaccessible with standard illumination schemes [START_REF] Gustafsson | Surpassing the lateral resolution limit by a factor of two using structured illumination microscopy[END_REF]. Contrary to standard laser scanning modalities like STED/RESOLFT, SIM allows acquisition of large field of view in limited times. However, SIM routinely provides only a ~2 fold resolution enhancement of standard widefield microscopy as compared to other super-resolution methods [START_REF] Gustafsson | Surpassing the lateral resolution limit by a factor of two using structured illumination microscopy[END_REF]. Non-linear saturated SIM using fluorophore saturation or photoswitchable proteins as in RESOLFT can achieve higher resolution enhancement (~50 nm), but requires an increased number of image acquisitions (up to 63) and complex reconstruction process [START_REF] Gustafsson | Nonlinear structured-illumination microscopy: wide-field fluorescence imaging with theoretically unlimited resolution[END_REF][START_REF] Rego | Nonlinear structured-illumination microscopy with a photoswitchable protein reveals cellular structures at 50-nm resolution[END_REF]. SIM was demonstrated for long-term live-cell imaging such as microtubules and other dynamic structures [START_REF] Rego | Nonlinear structured-illumination microscopy with a photoswitchable protein reveals cellular structures at 50-nm resolution[END_REF][START_REF] York | Resolution doubling in live, multicellular organisms via multifocal structured illumination microscopy[END_REF][START_REF] Olshausen | Superresolution Imaging of Dynamic MreB Filaments in B. subtilis-A Multiple-Motor-Driven Transport?[END_REF]. Three dimensional SIM imaging has been further achieved using 15 different pattern acquisitions per axial planes for reconstruction instead of 9 images to reject the out-of-focus light [START_REF] Shao | Super-resolution 3D microscopy of live whole cells using structured illumination[END_REF].

Additionally, whole cell volume imaging was achieved using 3D SIM in two colors [START_REF] Fiolka | Time-lapse two-color 3D imaging of live cells with doubled resolution using structured illumination[END_REF].

Interestingly, fast SIM imaging (11 Hz) was also developed with a 100 nm resolution for small field of view (~8x8 µm 2 ) [START_REF] Kner | Superresolution video microscopy of live cells by structured illumination[END_REF].

Single-molecule localization microscopy approaches.

It is well known that the position of isolated single fluorescent emitters can be determined by image analysis with greater precision than the diffraction limit. This feature that has been used for more than twenty years in live cell single particle/molecule studies [START_REF] Cognet | Advances in live-cell single-particle tracking and dynamic super-resolution imaging[END_REF], is key to provide nowadays super-resolved images. Super-resolution methods based on single-molecule localizations simply consist in reconstructing an image from single molecules localizations retrieved from a large number of movie frames (typically thousands of camera frames). The main requirement is that each frame contains the detection of well spatially separated fluorescent emitters [START_REF] Betzig | Proposed method for molecular optical imaging[END_REF]. Because, a large amount of single molecule detections has to be registered in order to reconstruct a high content super-resolved image, this acquisition process in inherently slow (typically more than a few seconds). We describe below three families of approaches considering how fluorescent molecules are stochastically isolated from non-fluorescent ones in each camera frame.

Photo-activation localization microscopy (PALM). The development of Photo-Activation

Localization Microscopy (PALM) [START_REF] Betzig | Imaging intracellular fluorescent proteins at nanometer resolution[END_REF][START_REF] Hess | Ultra-high resolution imaging by fluorescence photoactivation localization microscopy[END_REF] is closely linked to the advent of photo-activable proteins( 16) which allow to control by light the density of fluorescing proteins in each image.

Because PALM takes advantage of the versatility and specificity of genetically encoded fluorescently tagged molecules in cells, it has quickly become the tool of choice for superresolution live cell imaging. It is however restricted to expression systems which prevents studying endogenous proteins in their native environment. Interestingly, PALM is by design not restricted to biomolecules present at the cell plasma membrane and allows the study of intracellular biomolecules. In addition, by tracking the movement of each individual protein, PALM allows measuring local diffusion properties in living cells on short time scales [START_REF] Manley | High-density mapping of single-molecule trajectories with photoactivated localization microscopy[END_REF][START_REF] Rossier | Integrins beta1 and beta3 exhibit distinct dynamic nanoscale organizations inside focal adhesions[END_REF][START_REF] Gudheti | Actin mediates the nanoscale membrane organization of the clustered membrane protein influenza hemagglutinin[END_REF] and cellular structural changes in 3D on longer time scales [START_REF] Fu | In vivo structure of the E. coli FtsZ-ring revealed by photoactivated localization microscopy (PALM)[END_REF]. [START_REF] Rust | Sub-diffraction-limit imaging by stochastic optical reconstruction microscopy (STORM)[END_REF] and direct STORM (dSTORM) [START_REF] Wombacher | Live-cell super-resolution imaging with trimethoprim conjugates[END_REF] use switchable organic fluorophores placed in specific buffers (e.g. with reducing properties) instead of using fluorescent proteins as in PALM. Those probes can be targeted on genetically encoded or endogenous proteins using adequate ligands. STORM was first demonstrated using Cy3-Cy5 pairs(34) but was quickly extended in dSTORM to other synthetic fluorophores that can be stochastically and reversibly switched in the imaging buffers [START_REF] Heilemann | Super-resolution imaging with small organic fluorophores[END_REF][START_REF] Schermelleh | A guide to super-resolution fluorescence microscopy[END_REF]. STORM is particularly powerful for fixed cells applications [START_REF] Huang | Three-dimensional superresolution imaging by stochastic optical reconstruction microscopy[END_REF][START_REF] Dani | Superresolution imaging of chemical synapses in the brain[END_REF][START_REF] Loschberger | Super-resolution imaging visualizes the eightfold symmetry of gp210 proteins around the nuclear pore complex and resolves the central channel with nanometer resolution[END_REF] and can be extended in 3D [START_REF] Huang | Three-dimensional superresolution imaging by stochastic optical reconstruction microscopy[END_REF]. Yet, live cell experiments should be performed with great care due to possible issues with the use of reducing/oxidizing buffers that can affect cell integrity [START_REF] Jones | Fast, three-dimensional superresolution imaging of live cells[END_REF].

Stochastic optical reconstruction microscopy (STORM) and Ground state depletion microscopy followed by individual molecular return (GSDIM). STORM

Interestingly, it was shown that STORM can take advantage of some reducing thiol glutathione, naturally present at millimolar concentrations in bacteria [START_REF] Wang | Chromosome organization by a nucleoid-associated protein in live bacteria[END_REF] or in specific cell compartments like the nuclei of eukaryote cells using buffers with low cellular toxicity [START_REF] Klein | Live-cell dSTORM with SNAP-tag fusion proteins[END_REF][START_REF] Benke | Live-cell dSTORM of cellular DNA based on direct DNA labeling[END_REF]. Organelles from live cells, like the membrane and mitochondria, were also investigated using multicolor STORM [START_REF] Shim | Super-resolution fluorescence imaging of organelles in live cells with photoswitchable membrane probes[END_REF][START_REF] Benke | Multicolor single molecule tracking of stochastically active synthetic dyes[END_REF][START_REF] Wilmes | Triple-color super-resolution imaging of live cells: resolving submicroscopic receptor organization in the plasma membrane[END_REF]. Analogously to dSTORM, where stochastic photo-switching is used to control the number of emitting fluorophores, GSDIM is another acronym that covers the techniques employing the transition between the fluorescent singlet state and a metastable triplet state as the stochastic on-off switch (48 , 49). More precisely, efficient transition to the long-lived triplet state is achieved in such techniques by using high excitation intensities and similar imaging buffer as in STORM to allow obtaining triplet lifetime long enough to leave only a few emitting fluorophore at any time in each image. Under these conditions, GSDIM has been employed for imaging living cells using both fluorescent protein tags (as in PALM) or various organic fluorophores (as in STORM) that selectively bind to tagged proteins [START_REF] Lalkens | MRT letter: Nanoscopy of protein colocalization in living cells by STED and GSDIM[END_REF][START_REF] Lukinavicius | A near-infrared fluorophore for live-cell super-resolution microscopy of cellular proteins[END_REF].

Universal point accumulation imaging in the nanoscale topography (uPAINT).

In contrast with PALM, STORM and GSDIM that are based on the emitters stochastic photoswitching, the Universal Point Accumulation Imaging in the Nanoscale Topography (uPAINT)(52-55) method captures real-time molecular interactions to control the density of fluorescent emitters suitable for single molecule identification in each image. In the uPAINT approach, target molecules are individually imaged when a specific ligand coupled to a fluorescent dye binds to the target molecule. Indeed, unbound ligands are freely diffusing in the imaging buffer (with typical diffusion constants of the order of tens of µm 2 /s) and are not efficiently excited due to an oblique illumination excitation. The unbound ligands are, thus, not detected efficiently by the detector operating at typically video rate on the contrary to bound ligands, which diffuse together with a membrane receptor (with typical diffusion constants of less than 1 µm 2 /s) in the oblique laser illumination. With uPAINT, any binding entity conjugated to fluorophores having high specificity toward a target molecule (e.g. natural/synthetic ligand, antibody) can be used as fluorescent probes to reveal the targeted molecules. Applications included receptors and GPIanchored proteins diffusing on live cell membranes labeled with antibodies or synthetic ligands.

More interesting, it was also applied to image and track endogenous receptors at high densities like glutamate receptors in neurons [START_REF] Giannone | Dynamic superresolution imaging of endogenous proteins on living cells at ultra-high density[END_REF] and epidermal growth factor receptors (EGFRs) in culture living cells [START_REF] Winckler | Identification and super-resolution imaging of ligand-activated receptor dimers in live cells[END_REF]. Comparison between transfected non-endogenous receptors and endogenous glutamate receptors was also performed [START_REF] Nair | Super-resolution imaging reveals that AMPA receptors inside synapses are dynamically organized in nanodomains regulated by PSD95[END_REF]. A similar approach allowed tracking and imaging by continuously labeling sodium ion channels in live cells, using small fluorescently labeled molecule agents that display reversible binding to sodium ion channel [START_REF] Ondrus | Fluorescent saxitoxins for live cell imaging of single voltage-gated sodium ion channels beyond the optical diffraction limit[END_REF]. Interestingly, combining single molecule FRET and dual-color uPAINT allowed the specific super-resolution imaging and tracking of interacting receptors activated by their cognate ligand in live cells [START_REF] Winckler | Identification and super-resolution imaging of ligand-activated receptor dimers in live cells[END_REF]. This feature stems from the fact that in this uPAINT study, fluorescently tagged ligands are directly used as imaging probes, allowing to extract and image in real time the population of activated functional receptors upon ligand binding.

Discussion

Dynamics and resolution in live cells. In live cells or organisms, supramolecular structures and organelles morph in relatively short time scales often in reaction to stimuli (seconds to minutes and up to hours). To fully understand cell signaling that induce those processes, it is however important to investigate therein the dynamics of molecular (re)organizations. In particular, in order to grasp the full spectrum of mobility behaviors of biomolecules (up to 1 µm 2 /s for membrane receptors) fast video rate acquisition is required (20-100 Hz) on large fields of view.

Yet, in all super-resolution methods, breaking the diffraction limit on a given field of view comes at the expense of the acquisition time.

In point scanning RESOLFT/STED methods, a compromise between imaging large fields of view and fast acquisition speed has to be made because of the requirement for a dense pixilation.

Imaging rates in RESOLFT are rather slow because pixel integration times are limited by the protein photoswitching processes. Being based on stimulated emission, STED is not subject to this fundamental limit. However, increased resolution being achieved with high laser powers, care should be taken to ensure live cell integrity. RESOLFT/STED are able to resolve the movements of the slow structures (typ. 10-30 seconds for 512x512 pixels) like for instance, microtubule networks organizations [START_REF] York | Resolution doubling in live, multicellular organisms via multifocal structured illumination microscopy[END_REF][START_REF] Chmyrov | Nanoscopy with more than 100,000 'doughnuts[END_REF] and neuron morphology dynamics [START_REF] Rochefort | Dendritic spines: from structure to in vivo function[END_REF][START_REF] Berning | Nanoscopy in a living mouse brain[END_REF], both in cell cultures and live animals. Interestingly, by acquiring small fields of views, the dynamics of nanoscale structures can be monitored with higher imaging rates (28 Hz for 60x82 pixels) [START_REF] Lauterbach | Comparing video-rate STED nanoscopy and confocal microscopy of living neurons[END_REF].

Recently, large parallelization of the depletion patterns combined with the use matrix detectors drastically increased imaging speed over large field of views in RESOLFT [START_REF] Chmyrov | Nanoscopy with more than 100,000 'doughnuts[END_REF] and STED [START_REF] Yang | Large parallelization of STED nanoscopy using optical lattices[END_REF].

In its standard form, SIM uses 9 widefield fluorescent images to build a super-resolved image in typically one second. Many applications of SIM were described for imaging subcellular structures in living cells [START_REF] Kner | Superresolution video microscopy of live cells by structured illumination[END_REF][START_REF] Olshausen | Superresolution Imaging of Dynamic MreB Filaments in B. subtilis-A Multiple-Motor-Driven Transport?[END_REF][START_REF] Fiolka | Time-lapse two-color 3D imaging of live cells with doubled resolution using structured illumination[END_REF]. With saturated SIM (the structured illumination approach giving better spatial resolution), additional illumination patterns are needed for the reconstruction(21) leading to degraded time resolution. Importantly, in SIM techniques any aberration, sample movement or fluorophore photobleaching during the image sequence will induce artifacts that will strongly affect the quality of reconstructed super-resolved images [START_REF] Kner | Superresolution video microscopy of live cells by structured illumination[END_REF].

In single-molecule localization approaches two time-scales are relevant: (i) the raw-images acquisition rate which sets the individual molecules tracking time resolution (1-10's of ms). This rate also sets the single molecule pointing accuracy through its impact on the signal to noise ratio of each molecular detection [START_REF] Bobroff | Position measurement with a resolution and noise-limited instrument[END_REF][START_REF] Thompson | Precise nanometer localization analysis for individual fluorescent probes[END_REF]. The analysis of single-molecule trajectories provides local mobility maps on live cell regions with high spatiotemporal resolutions (30-32, 54, 57). (ii) the second time scale is set by the total number of images needed to reconstruct a super-resolved image. Indeed, in addition to the pointing accuracy, the local density of single molecule detections obtained from a studied structure also plays a central role in the final spatial resolution (as enounced by the Nyquist theorem). For instance, in order to obtain images with 10 nm resolution, local densities of at least 10,000 detection/µm 2 are needed. Hence, thousands of image frames are commonly acquired justifying global recording times of the order of seconds to minutes. This time scale directly defines the time resolution at which nanoscale organization of molecular assemblies (e.g. cellular organelles) can be analyzed.

Computer analysis requirements.

Conversely to RESOLFT/STED methods, that do not require any post-acquisition analysis, the main source of complexity of SIM is its sophisticated algorithms required for image reconstruction. Similarly for single-molecule localization based techniques, the position of the emitting single molecules has to be retrieved using cutting-edge software as described in a recent comparative study [START_REF] Chenouard | Objective comparison of particle tracking methods[END_REF].

Labeling strategies and consequences. Expression systems in concert with fluorescent protein engineering provide a method of choice to study with high-specificity sub-cellular organizations in live-cell making of RESOLFT, SIM and PALM essential methods for the applications described in this review. However, despite their wide applicability, one should bear in mind that some signaling and structural artifacts can arise due to the use of fluorescent proteins [START_REF] Swulius | The helical MreB cytoskeleton in Escherichia coli MC1000/pLE7 is an artifact of the N-Terminal yellow fluorescent protein tag[END_REF]. In this context, SIM, uPAINT, STED and to some extent STORM/GSDIM, using fluorophores conjugated to specific ligands should be considered as they are compatible with studying endogenous receptors in living cells. Interestingly, by synchronizing single molecule detection and ligand-induced receptor activation, uPAINT is to-date the only super-resolution method allowing studying in real-time specifically activated functional receptors and their interactions at the membrane of living cells [START_REF] Winckler | Identification and super-resolution imaging of ligand-activated receptor dimers in live cells[END_REF]. Noteworthy, protein number quantification can in principle be performed using PALM and uPAINT since in these methods, photobleaching irreversibly turns off fluorophores after their detection. In STORM/GSDIM however, reversible stochastic switching of fluorophores can bias such quantitative analysis because observing the same fluorescent molecules more than once is plausible. Interestingly, recent advances in self labeling proteins such as SNAP, CLIP and Halo tags allow efficient live cell protein labeling including intracellular ones [START_REF] Benke | Multicolor single molecule tracking of stochastically active synthetic dyes[END_REF][START_REF] Lukinavicius | A near-infrared fluorophore for live-cell super-resolution microscopy of cellular proteins[END_REF][START_REF] Stagge | SNAP-, CLIPand Halo-tag labelling of budding yeast cells[END_REF] and provide valuable tools to perform multi-color super-resolution imaging that can be in principle applied to STED, STORM or uPAINT. Detecting molecules using two objectives [START_REF] Juette | Three-dimensional sub-100 nm resolution fluorescence microscopy of thick samples[END_REF][START_REF] Xu | Dual-objective STORM reveals threedimensional filament organization in the actin cytoskeleton[END_REF], by moving the sample in the axial direction [START_REF] Chasles | Full-field optical sectioning and three-dimensional localization of fluorescent particles using focal plane modulation[END_REF] or by interfering the signals obtained from two objectives(73-74) could also yield similar resolution improvements along the optical axis. Although, the last approach is the most precise (~20 nm), it is also the most complex to implement. Finally, STED/RESOLFT can also be extended in 3D by scanning distinct axial planes independently [START_REF] Tonnesen | Spine neck plasticity regulates compartmentalization of synapses[END_REF]. Summary Super-resolution approaches described in this mini-review were proven to grasp information on subcellular organization at different time scales using various labeling probes. .

Studying structures in three dimensions

Conclusion

In the last two decades, super-resolution approaches have provided new insights into sub-cellular organization at nanometer scale resolutions. Several of these methods have reached a level of maturity which makes them routinely applicable to many biological systems. For live cell imaging, each approach has however its own advantages and drawbacks as described in this mini-review. In this context, combining these techniques can be valuable to tackle specific biological questions taking benefit of each method like for instance, to ensure orthogonal labeling for multicolor imaging [START_REF] Wilmes | Triple-color super-resolution imaging of live cells: resolving submicroscopic receptor organization in the plasma membrane[END_REF]. Interestingly other recent approaches can also be implemented to increase the image resolution by a factor two to four, by applying software analytical tools to standard experimental imaging data sets. These include deconvolution microscopy [START_REF] Sibarita | Deconvolution microscopy[END_REF], Stochastic Optical Fluctuation Imaging (SOFI) [START_REF] Dertinger | Fast, background-free, 3D super-resolution optical fluctuation imaging (SOFI)[END_REF], Bayesian localization microscopy [START_REF] Cox | Bayesian localization microscopy reveals nanoscale podosome dynamics[END_REF] and compressed sensing [START_REF] Studer | Compressive fluorescence microscopy for biological and hyperspectral imaging[END_REF][START_REF] Zhu | Faster STORM using compressed sensing[END_REF][START_REF] Zanella | Towards real-time image deconvolution: application to confocal and STED microscopy[END_REF]. Furthermore, we foresee that correlating the information gathered by such specific molecular imaging methods with structural information obtained by electron microscopy will provide new insights on molecular subcellular organization and interaction.

Figure 1 :

 1 Figure 1: Schematic description of the super-resolution microscopy approaches. All images for this didactic description are computer generated. Object to be imaged consisting of fluorescent emitters (A) and corresponding diffraction limited image (B). (C) In RESOLFT/STED, a focused excitation beam (Cyan) superimposed with a doughnut shaped depletion beam (Red) are scanned over the sample to acquire an image at high resolution (down to ~50-80 nm in live cells). (D) In SIM, multiple wide-field images are acquired using sinusoidal illumination grid patterns to obtain, after software reconstruction, high-resolution images (down to ~50-100 nm in live cells using non-linear saturated illumination). (E) In single molecule localization microscopy, a large number of wide-field images containing a few isolated single fluorescent emitters are successively acquired. A high-resolution image is reconstructed from the localizations of each individual molecule. Resolutions down to ~50 nm are commonly achieved in live cells. In the example provided, we considered the detection of 80% of the molecules present in the object image. Scale bar represents

Figure 2 :

 2 Figure 2: Examples of achievements obtained with super-resolution microscopy in live biological samples. (A) SIM: Total-internal reflection microscopy image series of eGFP-α-tubulin in a live S2 cell and corresponding SIM images revealing the elongation followed by a rapid shrinking of a microtubule. Integration time of 270 ms per frame. This figure was adapted from(18). (B) STED: Continuous wave STED images of the yellow fluorescent protein citrine targeted to the endoplasmic reticulum in live cells revealing small tubules (~60 nm). Image sequences show morphing of the endoplasmic reticulum at arrows (pixel size= 20 nm, 10 s recording time per image). Scale bar= 1 µm. This figure was adapted from(6). (C) PALM: numerous single trajectories of β3-integrin fused with mEOS2, obtained on a single MEF cell with PALM, revealing that β3-integrin undergo slower free-diffusion inside focal adhesions (grey) than outside as well as confined diffusion and immobilization. Figure adapted from(31).(D) STORM: Spatial dynamics of cortical actin skeleton stained with Lifeact-HaloTag/ATTO655. Each reconstruction was obtained using 1000 frames (2 ms per frame). Scale bar= 1 µm. This figure was adapted from[START_REF] Wilmes | Triple-color super-resolution imaging of live cells: resolving submicroscopic receptor organization in the plasma membrane[END_REF].

(

  E) uPAINT: Live cell super-resolution imaging of membrane epidermal growth factor receptor (EGFR) dimers based on single-molecule FRET induced by fluorescent ligand activation. Inset highlights preferential cell edge localization of EGFR dimers. In addition, uPAINT provides numerous single molecule trajectories on a single cell allowing extracting the diffusion properties of the EGFR dimer population from the whole ligand activated EGFR population. This figure was adapted from(56).

.

  Several methods were developed to improve axial resolution in fluorescence imaging. The most widely used strategies are based on singlemolecule localization and provide axial information (~20-70 nm) of the position without severely altering radial nor time precision. By shaping the detection point-spread function along the axial position, single molecule position can be precisely determined along the optical axis(38, 68-69).

Table 1

 1 summarizes the main pros/cons of the approaches discussed here and outlines the different spatiotemporal fundamental limits.

	Single-molecule approaches

Table 1 :

 1 Table comparing the super-resolution approaches presented in this review. ** Using non-linear saturated SIM allows accuracy enhancement up to 50nm(21).

* Parallelization of STED nanoscopy using optical lattices was recently achieved with an imaging rate of 12.5 Hz for a 2.9µmx2.9µm image(5). It requires a simple reconstruction algorithm. ** Large parallelization of the depletion patterns drastically imaging speed over larger field of views in RESOLFT (120µmx100µm in ~3 seconds(59)). It requires a simple reconstruction algorithm. *

Acknowledgments

We acknowledge financial support from the Fondation pour la Recherche Medicale (A.G.G.), Agence Nationale de la Recherche, Region Aquitaine, the French Ministry of Education and Research, the CNRS, the European Research Council and FranceBioImaging (Grant N° ANR-10-INSB-04-01).


Chinh Pham The

Tuyet Anh Dang

Quoc Thi Phuong Hoang

Duy Anh Ngo

Tien Doan

Thu Ha

Nguyen Thi

Tham Pham

Vu Thi

Mickael Jean

Pierre Van De Weghe

Anh Quoc

Ngo

Duy Tien Doan

Thu Ha Nguyen

Tuyen Nguyen Van

Synthesis of new bioisosteric hemiasterlin analogues with extremely high cytotoxicity

des établissements d'enseignement et de recherche

Hemiasterlins belong to a family of naturally occurring tripeptides from marine sponges. 1 The important derivatives of hemiasterlins are hemiasterlin A, hemiasterlin B, and hemiasterlin C, which were isolated from marine sponge Auletta and Cymbastella (Fig. 1) and exhibited potent cytotoxicity in vitro against murine leukemia P388 and human breast, ovarian, colon, and lung cancer cell lines. 2,3 Hemiasterlins suppress microtubule depolymerization presumably by binding to the vinca-alkaloid binding-domain of tubulin and leading to mitotic arrest and cell death. 4a The synthetic analog HTI-286 (2) displayed especially potent cytotoxicity against paclitaxel (Taxol TM ) resistant turmor cell lines in vitro and in vivo and is currently in clinical trials. 4b There are several reports on the synthesis of new derivatives of hemiasterlin in which the indole aromatic ring in the moiety A of the original molecule was replaced by aryl functional groups. 5,6a However, asymmetric synthesis of the stereospecific amine group and especially the gem-dimethyl moiety were proved to be highly problematic. To overcome this difficulty, several studies explored modifications of segment A in which the gem-dimethyl moiety has been eliminated. Some of these derivatives showed promising cytotoxic activity. 6 In recent years, previous works have shown that the presence of a α,β-unsaturated carbonyl system in peptide derivatives can improve the biological activity compared to the initial compound. 7,8 Explanations may include either a limitation of free energy of the peptide by addition of a constrained system or by an electrophilic character of the compound leading to the possibility to form covalent bond with the protein target. Based on the idea that hemiasterlin derivatives containing α,β-unsaturated carbonyl systems could induce a remarkable cytotoxicity, we decided to synthesize new hemiasterlin derivatives in which the α,αdimethylbenzylic group (fragment A) is replaced by a α,β-unsaturated carbonyl group.

N OH O N H O R 2 O NH N R 1 A B C N OH O N H O O NH R 2 (HTI-286 R = H) 3 (HTI-286 analog R = OMe) Hemiasterlin (1) Hemiasterlin A Hemiasterlin B Hemiasterlin C R 1 = Me R 1 = H R 1 = H R 1 = Me R 2 = Me R 2 = Me R 2 = H R 2 = H
New hemiasterlin derivatives were synthesized via classical peptide coupling reactions between two fragments 7a-h and 14a,b. A general procedure for the synthesis of compound 7 is outlined in Scheme 1. Compounds 7 were prepared from glycine in three steps. 9 The synthesis started by acetylation of glycine with acetic anhydride in water at room temperature following by condensation with aryl aldehydes using sodium acetate in the presence of acetic anhydride at 90 °C for 12 h which afforded azalactones 6a-h in 72-83% yields. 9 Finally, azalactones 6a-h were hydrolyzed in aqueous sodium hydroxide, followed by treatment with hydrochloric acid (12 N) at 100 °C for 4 h to give compounds 7a-h in 65-88% yields.

18a R=H (73%) 18b R=Me (74%) R R 19a R=H (92%) 19b R=Me (91%) CF 3 COO CF 3 COO O O (i) (ii) 15a R=H (78%) 15b R=Me (65%) (iv) (v) (vi) 13a R=H 13b R=Me (iii)
Scheme 2. Reagents and conditions (i) 1.1 equiv NH(Me)OMe, 1.1 equiv EDC, 1.1 equiv HOBt, 2.0 equiv i-PrNHEt, DMF, rt, 12 h. (ii) 4.0 equiv LAH, THF, rt, 1 h. (iii) Ph3P=C(CH3)CO2Et, CH2Cl2, reflux, 6 h. (iv) TFA, CH2Cl2, rt, 1 h. (v) 1.0 equiv Boc-L-Leucine, 1.1 equiv EDC, 1.1 equiv HOBt, 2.0 equiv i-PrNHEt, DMF, rt, 12 h. (vi) TFA, CH2Cl2, rt, 1 h.

The final compounds 16a-h were then prepared in two steps after peptide coupling reactions of 14a with amides 7a-h following by the saponification of ester using a 1 N lithium hydroxide solution (Scheme 3-a). Similarly, the hemiasterlin derivatives 18a-g were obtained from 14b (Scheme 3-b).

H N CO 2 Et N H NHAc Ar 19a H N CO 2 H N H NHAc Ar 20a, 60% -20b, 50% -20c, 55% 20d, 75% -20e, 70% -20f, 65% 20g, 71% -20h, 60% 21a, 90% -21b, 94% -21c, 90% 21d, 98% -21e, 90% -21f, 95% 21g, 91% -21h, 93% N CO 2 Et N H NHAc Ar 19b N CO 2 H N H NHAc Ar 22a, 70% -22b, 73% -22c, 67% 22d, 86% -22e, 65% -22f, 68% 22g, 70% 23a, 94% -23b, 98% -23c, 92% 23d, 91% -23e, 92% -23f, 90% 23g, 93% (a) (b)
O O O O O O O O (i) (i) (ii) (ii)
Scheme 3. Reagents and conditions (i) 1.0 equiv 12a-h, 1.1 equiv EDC, 1.1 equiv HOBt, 2.0 equiv i-PrNHEt, DMF, rt, 12 h. (ii) 10 equiv LiOH, MeOH : H2O (2:1), rt, 10 h.

All compounds 15a-h, 16a-h, 17a-g and 18a-g were evaluated in vitro for their cytotoxic activity against four human tumor cell lines (KB, Hep-G2, LU and MCF7) and the results were summarized in Table 1. Eleven hemiasterlin derivatives showed strong activity against the KB, Hep-G2 cell line with IC50 value below 100 nM. Analogs 16b, 17b and 18f exhibited potent cytotoxicity against the KB cell line with IC50 = 8.2, 3.5 and 3.7 nM, respectively. Meanwhile analogs 16b, 2217c and 17f displayed potent cytotoxicity against the Hep-G2 cell line with IC50 value 17.8, 16.3 and 3.7 nM, respectively. Derivatives 15a, 15e and 17a presented a cytotoxic activity against MCF7 cell line with IC50 value 42.3, 60.0 and 67.9 nM. Concerning the last cell line, the LU cell line, the hemiasterlin analogs showed weak activities with IC50 values above 269 nM. It is noteworthy to mention that two derivatives 17a and 17f present a cytotoxic activity against two cancer cell lines (KB, Hep-G2) comparable with those of ellipticine and paclitaxel In conclusion, a concise synthetic approach for new modified hemiasterlin derivatives was achieved in which the α,α-dimethylbenzylic group and amino NHMe moiety were replaced respectively by a α,β-unsaturated aryl and an amide NH-Ac group avoiding the synthesis of the chiral fragment A. Most of these derivatives possess strong cytotoxic activity on three human tumor cell lines (KB, Hep-G2 and MCF7) and two analogs, 17b and 17f, showed a comparable cytotoxicity activity to paclitaxel and ellipticine against KB and Hep-G2 cancer cell lines. Based on previously reported work, 6b we can envisage that our hemiasterlin derivatives act as tubulin polymerization inhibitors. From our best compounds, more detailed biological studies will be undertaken and will be reported in due course.

Figure 1 .

 1 Figure 1. Structures of hemiasterlin derivatives and HTI analogs.

11a

  Ar = naphthan-2-yl 11b Ar = furan-2-yl 11c Ar = furan-3-yl 11d Ar = thiophen-3-yl 11e Ar = 3-methoxyphenyl 11f Ar = 4-methoxyphenyl 11g Ar = 4-bromophenyl 11h Ar = 3-bromophenyl

  PrNHEt) at room temperature for 12 h.10 Reduction of 9a,b using LiAlH4 in THF was carried out at room temperature for 1 h to give aldehydes 10a,b in 78% and 65% yields, respectively.11 Afterward, Wittig reaction of aldehydes 10a,b with ethyl 2-(triphenylphosphoranylidene)propanoate was carried out at reflux in CH2Cl2 for 6 h to afford the alkenoates 11a and 11b in 86% and 88% yields, respectively.6,12 Then, removal of the Boc group using trifluoroacetic acid in CH2Cl2 at room temperature for 1 h led to 12a,b in high yields (90 and 95%). The expected compounds 14a,b are finally obtained after coupling 12a,b and Boc-L-leucine in the presence of EDC/HOBt in DMF at room temperature for 12 h following by Boc deprotection in classical reaction conditions.

		R	Boc N	CO 2 H		R	Boc N	O	N	Me	R	Boc N	O	H	R	Boc N	CO 2 Et
											OMe
							14a R=H (86%)				16a R=H (86%)
							14b R=Me (81%)				16b R=Me (88%)
	H 3 N		N			CO 2 Et			BocHN		N	CO 2 Et	R	H 2 N	CO 2 Et
																17a R=H (90%)
																17b R=Me (95%)
	H 2 N	CO 2 H	(i)	AcHN	CO 2 H	(ii)	Ar	N	O O			(70%) (73%) (73%) (72%)
													CH 3			(75%) (74%)
																(83%)
																(80%)
	(iii)	Ar		CO 2 H	12a, 82% -12b, 80% -12c, 75% 12d, 65% -12e, 85% -12f, 83%
			NHAc		12g, 88% -12h, 87%

Scheme 1. Reagents and conditions (i) 2.0 equiv Ac2O, H2O, rt, 20 h. (ii) 0.75 equiv ArCHO, 1.0 equiv AcONa, Ac2O, 90 °C, 12 h. (iii) NaOH (1 N) then HCl (12 N), 100 °C, 4 h. Compounds 14a,b were obtained from Boc-L-valine in 6 steps as depicted in Scheme 2.

4

In the first step, Boc-L-valines 8a,b were converted to Weinreb amide 9a,b in good yields (86 and 81%) by treatment with a mixture of N,O-dimethylhydroxylamine hydrochloride,

1-ethyl-3-(3- dimethylaminoproryl)

carbodiimide (EDC) and hydroxybenzotriazole (HOBt) in the presence of N-ethylisopropylamine (i-

Table 1 .

 1 Cytotoxicity evaluation.

				IC50 (nM)
	Entry compound	KB	Hep-G2	LU	MCF7
	1	15a	30.8	39.7	>269	42.3
	2	15b	160.5 214.2 >269 >269
	3	15c	86.2	67.3	>269 >269
	4	15d	107.4	57.6	>269	203
	5	15e	77.4	47.7	228.4	62
	6	15f	>269 >269 >269 >269
	7	15g	>269 >269 >269 >269
	8	15h	>269 >269 >269 >269
	9	6a	63	33.4	>269 >269
	10	6b	8.2	17.8	>269 >269
	11	6c	155.4 >269 >269 >269
	12	6d	>269 >269 >269 >269
	13	6e	>269 >269 >269 >269
	14	6f	>269 >269 >269 >269
	15	6g	>269 >269 >269 >269
	16	6h	>269 >269 >269 >269
	17	17a	23.0	23.1	>269	67.9
	18	17b	3.5	31.4	231.0 178.4
	19	17c	24.1	16.3	158	112.8
	20	17d	202	15.0	>269 >269
	21	17e	>269 >269 >269 >269
	22	17f	3.7	3.7	>269	215
	23	17g	49.0	>269 >269 >269
	24	18a	69.7	13.0	>269 >269
	25	18b	188.0 >269 >269 >269
	26	18c	>269 149.4 >269 >269
	27	18d	234	63.2	>269 >269
	28	18e	>269 >269 >269 >269
	29	18f	15.8	63.8	>269 >269
	30	18g	>269 >269 >269 >269
	31	Ellipticine 1.26	1.26	1.82	2.15
	32	Paclitaxel	3.9	0.19	-	-

Acknowledgments

This work was financially supported in part by scientific research and technological development project (code: ĐT.NCCB-ĐHUD.2011-G/07).

Supplementary Material

Supplementary data associated with this article can be found, in the online version, at doi: