Leam Sreng

Brice Temime-Roussel

Henri Wortham

Christiane Mourre

Chemical identification of 'maternal signature odours' in rat

Keywords: odour preference, maternal signature, learning and memory, amniotic fluid, diet, rodents

Newborn altricial mammals need just after birth to locate their mother's nipples for suckling. In this precocious behaviour, including for the human baby, maternal odour via the olfactory process plays a major role. Maternal odour emitted by lactating females or by amniotic fluid (AF) attracts pups, but the chemical identity of this attractant has not yet been elucidated. Here, using behavioural tests and gas chromatography coupled with mass spectrometry (GC-MS) techniques, we show that AF extracts from rat pregnant female, nipples, ventral skin, milk and nest extracts of mother contained 3-6 active substances. AF extracts contained 3 active compounds: ethylbenzene, benzaldehyde and benzyl alcohol, and their mixture in similar proportions to those found in AF extracts, in a ratio, respectively, of 1:1:12 (700 ng), attracts pups as putative maternal attractant substances (MAS). These 3 AF substances have already been identified in milk, nipples, ventral wash and nest extracts of mother, but not in feces. Moreover, anethole flavour incorporated in pregnant rat and mother's diet is also detected in AF, nipples, milk and nest extracts and the pups are attracted to anethole odour, but not in the case of the no-anethole pups. Maternal attractant substances, combined with diet flavours present in the AF bath, represent olfactory signals as 'maternal signature odours' (MSO) that are learned by foetus and pups. These findings open the way to improved understanding of the neurobiology of early olfactory learning and of the importance of evolutionarily conserved survival behaviour in many mammal species.

Introduction

For the survival and adaptation process of the species, newborn mammals are confronted, shortly after birth, with the need to locate, grasp and suck from the nipple of their mother. This is particularly obvious for the neonate which at birth must be able to use and respond appropriately to a whole range of new stimuli. In this early adaptation, maternal odour, via the olfactory mechanisms, plays a major role in early behavioural development, such as orientation to maternal odour and suckling [START_REF] Cheal | Social olfaction: a review of the ontogeny of olfactory influences on vertebrate behavior[END_REF][START_REF] Rosenblatt | Olfaction mediated developmental transition in the altricial newborn of selected species of mammals[END_REF][START_REF] Varendi | Does the newborn baby find the nipple by smell?[END_REF][START_REF] Porter | Unique salience of maternal breast odors for newborn infants[END_REF][START_REF] Schaal | Mammary odor cues and pheromones: mammalian infant-directed communication about maternal state, mammae, and milk[END_REF][START_REF] Schaal | Chemical signals 'selected for' newborns in mammals[END_REF]. The role of olfactory signals in the mediation of mother-newborn interactions and precocious attachment has been demonstrated in several species of rodents [START_REF] Carter | Olfactory imprinting and age variables in the guinea-pig, Cavia porcellus[END_REF][START_REF] Noirot | Selective priming of maternal responses by auditory and olfactory cues from mouse pups[END_REF][START_REF] Schapiro | Behavioral response of infant rats to maternal odor[END_REF][START_REF] Gandelman | Olfactory bulb removal eliminates maternal behavior in the mouse[END_REF][START_REF] Leon | The development of the pheromonal bond in the albino rat[END_REF][START_REF] Moltz | Stimulus control of the maternal pheromone in the lactating rat[END_REF][START_REF] Wallace | The control and function of maternal scent marking the Mongolian gerbil[END_REF][START_REF] Devor | Attraction to home-cage odor in hamster pups: specificity and changes with age[END_REF][START_REF] Porter | Maternal pheromone in the spiny mouse (Acomys cahirinus)[END_REF][START_REF] Breen | Maternal pheromone: A demonstration of its existence in the mouse (Mus musculus)[END_REF][START_REF] Hudson | Pheromonal release of suckling in rabbits does not depend on the vomeronasal organ[END_REF]. In rat, olfaction is essential in early development [START_REF] Salas | Development of olfactory bulb discrimination between maternal and food odors[END_REF][START_REF] Gregory | Development of olfactory-guided behavior in infant rats[END_REF][START_REF] Singh | Effects of nasal ZnSO4 irrigation and olfactory bulbectomy on rat pups[END_REF].

Without maternal assistance, the neonate rat, although blind and deaf, can crawl under its mother's body, position itself, choose a free nipple, and suckle. Rat pups which have been rendered insensitive to olfactory cues by olfactory denervation with intranasal ZnS04 are unable to attach to the nipples of an anaesthetized lactating female and when housed with their own unanaesthetized mother, lose weight and often die [START_REF] Singh | Effects of nasal ZnSO4 irrigation and olfactory bulbectomy on rat pups[END_REF]. Rat pups are also highly dependent on odour cues from the dam's ventrum for nipple location and attachment [START_REF] Hofer | Evidence that maternal ventral skin substances promote suckling in infant rats[END_REF][START_REF] Teicher | Suckling in newborn rats: eliminated by nipple lavage, reinstated by pup saliva[END_REF]). An odorous attractive substance, released by a lactating female rat and impregnating a soiled litter [START_REF] Nyakas | Olfaction guided approaching behaviour of infantile rats to the mother in maze box[END_REF][START_REF] Salas | Development of olfactory bulb discrimination between maternal and food odors[END_REF][START_REF] Schapiro | Behavioral response of infant rats to maternal odor[END_REF][START_REF] Gregory | Development of olfactory-guided behavior in infant rats[END_REF][START_REF] Galef | Olfactory mediation of mother-young contact in Long-Evans rats[END_REF], as well as another substance called maternal pheromone [START_REF] Moltz | Stimulus control of the maternal pheromone in the lactating rat[END_REF], serves as a powerful attractant to the young and incites early walking in three-day-old neonatal rats [START_REF] Jamon | Early walking in the neonatal rat: a kinematic study[END_REF]. Moreover, the ventral zone of lactating mothers contains olfactory cues that orient rat pups to finding and attaching to nipples, but chemical washing of the nipple area eliminated this behaviour [START_REF] Hofer | Evidence that maternal ventral skin substances promote suckling in infant rats[END_REF][START_REF] Singh | Oxytocin reinstates maternal olfactory cues for nipple orientation and attachment in rat pups[END_REF]Blass and Teicher, 1980). The relationship of maternal ventral skin substances and maternal pheromone is not yet clear. The nature of these putative maternal attractant substances is as yet unknown. Do these olfactory signals performing different behavioural functions originate from the same substances?

Previous studies in many animal species showed that neonates are capable of perceiving and recognizing the odour profile of their amniotic fluid (AF). Prenatal exposure to odour substances introduced into the AF [START_REF] Pedersen | Prenatal and postnatal determinants of the 1st suckling episode in albino rats[END_REF] or ingested by their pregnant mother may influence responsiveness to those same cues after birth [START_REF] Hepper | The discrimination of human odour by the dog[END_REF][START_REF] Schaal | Olfaction in utero: can the rodent model be generalized?[END_REF][START_REF] Mennella | Prenatal and postnatal flavor learning by human infants[END_REF][START_REF] Wells | Prenatal olfactory learning in the domestic dog[END_REF]. Moreover, some newborn mammals are attracted to the odour of AF per se [START_REF] Schaal | Olfaction in utero: can the rodent model be generalized?[END_REF][START_REF] Logan | Learned recognition of maternal signature odors mediates the first suckling episode in mice[END_REF]. What are the respective roles of putative maternal attractant substances (MAS) and of odorant intake of AF?

The purpose of the present study is to assess the source and identify volatile active compounds of the 'maternal signature odours' (MSO) by bioassay and gas chromatography-mass spectrometry (GC-MS) techniques. We used direct nest contact and Y maze tests to assess attractive natural and synthetic substances with newborn rats and 12-14 day-old post-natal rats.

Moreover, a substance, anethole, was incorporated in pregnant and mother rats' diet to assess whether diet flavours could moreover generate a specific olfactory signal as in mice 'maternal signature odours' (MSO) [START_REF] Wyatt | Pheromones and signature mixtures: defining species-wide signals and variable cues for identity in both invertebrates and vertebrates[END_REF][START_REF] Wyatt | Pheromones and animal behavior. Chemical signals and signatures[END_REF][START_REF] Logan | Learned recognition of maternal signature odors mediates the first suckling episode in mice[END_REF]. Signature odours have been proposed to underlie kin recognition between ewe and lambs [START_REF] Wyatt | Pheromones and signature mixtures: defining species-wide signals and variable cues for identity in both invertebrates and vertebrates[END_REF][START_REF] Porter | Individual olfactory signatures as major determinants of early maternal discrimination in sheep[END_REF].

Previously described individual olfactory signatures act to enable a ewe to distinguish and identify her individual lambs [START_REF] Porter | Individual olfactory signatures as major determinants of early maternal discrimination in sheep[END_REF]). Here we show the presence of 3 active compounds of the MAS in AF, and the existence of chemosensory cues related to the mother's diet. These findings suggest that the foetus and pups learn and memorize a specific combination of volatile odours or 'maternal signature odours' (MSO), facilitating suckling just after birth.

Materials and methods

Animals and experimental design

Wistar rats (male, virgin female, gravid female and female with litters) (280-300 g) (Charles River Company, France) were housed with pine wood shavings and given food (SAFE, A04, route de St Bris, 89290 Augy, France) and water ad libitum at a constant temperature (22°C), under a 12h/12h light/dark cycle (lights on at 7:00 a.m.). Each gravid female was individually housed and the date of parturition (postnatal day 0, P0) was recorded. Male and female pups used were postnatal day 5 (P5) in direct nest contact tests and postnatal day 12-14 (P12-14) in Y maze tests.

The young rats were isolated from their mothers for at least 1 hour before the tests and put together in a box maintained at 24°C. Each pup was identified by a number marked on its back.

Pups were naïve to each stimulus and were tested only once for a given odour. For each test, pups came from 2-3 different litters. The behavioural tasks were completed in a single-blind design in an experimental room at 24°C. All experiments were carried out in accordance with the European Communities Council Directive (86/609/EEC). Care was taken to minimize the numbers of animals used and to maintain them in good general health. Aix-Marseille Université and the Centre National de la Recherche Scientifique (CNRS) approved the study.

Behavioural tests

Direct nest contact test. The test apparatus consisted of a white plastic pan (28 cm length x 18 cm width x 4 cm height), divided into two parts, and separated by about 1.5 cm midline space (Fig. 2A).

One side contained the nest wood shavings of lactating female (~35g) and the other side contained the nest wood shavings of virgin female or clean nest shavings. Pups (P5), placed in the midline space, reacted to one type of nest shaving by snout probing, moving the head, crawling, burying in the nest material to the left or right side. The pup behaviour was recorded on a digital video camera (Canon Power Shot A95). The choice of a nest shaving was made when pups stopped walking or moving but stayed in the same nest side at least 5 sec. The choice and amount of time each pup spent over each type of nest shavings was recorded within a 60-second trial time.

Y maze test

Apparatus. A Y maze olfactometer in plastic material was composed of a start box (20 cm length, 6 cm width, 4 cm height) and two choice arms, (12 cm length with removable transparent cover (135° angle between start box and choice arms, 90° angle between each choice arm)) (Fig. 2B). The end of each choice arm was delimited by a removable Plexiglas door with a hole (4 mm) in the middle. A stimulus box comprising a plastic cylinder tube (6 cm length, 2.6 cm diameter) was positioned near the end of each choice arm. The choice arm and stimulus box were connected by a Teflon tube (2 cm x 4 mm in diameter) for delivery of the air and volatile substances. At the other end of the stimulus box, that was removable, a Teflon tube was attached as an air flow entry. Air flow used was 480 ml/min. At the junction of the choice arms, a central wall in Plexiglas (4 cm x 4 cm) was placed 3 mm from the screen moving door. This wall enabled the two stimuli air streams to be conducted to the beginning of the start box where the pups were positioned.

Test procedure. The 3-min test begins when a pup is placed in the start box, in front of the removable Plexiglas door. The door is then opened, enabling the pup to access the odour stimuli and make a choice. When the pup's snout has passed through a limit line, traced in each choice arm, two cm behind the moving door of the start box, the choice was recorded. It was noted "+" when a stimulus was chosen. When pups did not pass through the line, a no-response was noted and the pup was excluded from the experiment. Every 5 tests, the stimuli were changed and placed in the opposite choice arms. The olfactometer was cleaned with detergent, rinsed with distilled water, then with 95 % ethanol and dried. The performance was noted by an observer blind to the conditions of the experiments (in particular the nature of the tested compounds).

Olfactory stimuli

Different crude materials, extracts (nipples, milk, AF, nest shavings, feces, diet), synthetic compounds (alone or in a mixture), and polar solvent as control were used as olfactory stimulus.

Crude materials -The stimulus boxes were filled with clean litter, feces or with nest shavings of gravid, lactating or virgin female (34 ml), free of feces. For the crude amniotic fluid (AF), a filter paper was impregnated with fluid (50 µl), put in the stimuli box and clean filter paper was used as control.

Extracts -To determine the composition of the maternal chemosensory cues, we used an aprotic polar solvent (dichloromethane) because most of the substances of biofluids are hydrophilic, as in the AF and milk. Moreover, the polar solvent is able to extract polar and apolar molecules as fatty acids.

Organ and tissue extracts (nipples, milk, skin, and amniotic fluid).

Three pregnant (18 days of gestation) and three lactating females (P4) of normal and anethole groups were anaesthetized with ketamine (5%, Virbac, France) and medetomidine (1 mg/ml, Janssen), injected subcutaneously (0.33 ml/kg). 500 µl milk were collected from the lactating females, pooled in a cooled vial and extracted in 1 ml solvents. The nipples of gravid and lactating rats with the addition of surrounding skin (approximately 2 mm 2 ) and the skin from the mother's sides (approximately 4 mm 2 ) were removed and soaked in 3 ml of dichloromethane during 1 hr, then the skin and nipples were removed from the solvents. Amniotic fluid was collected from amniotic cavities and transferred to a vial at 4°C in a pooled sample and extracted in polar solvent in the ratio of 500 µl of AF for 1 ml of solvent during 1 hr. Then, the milk and AF extracts were vortexed for 1 min, centrifuged (10,000 tr/min) for 5 min and only the solvent parts were removed.

All extracts were evaporated to 0.5 ml with nitrogen stream, stored in the refrigerator at -20° C until used for behavioural tests or for GC-MS analysis.

Ventral wash extracts.

Each nipple and ventral surrounding tissue of three anaesthetized lactating females were washed 3 times with cotton swabs impregnated with polar solvent. Each cotton swab was transferred to a vial containing 3 ml of solvent and soaked for 1 hr. Then the cotton swab was removed from the solvent and the extracts were evaporated to a final 0.5 ml with nitrogen stream and stored in a freezer at -20°C prior to use.

Diet and feces extracts. 200 mg of broken chow or of feces were infused in 500 µl polar solvent for 30 min, centrifuged (10,000 tr/min) for 5 nm and only the solvent parts were removed. These extracts were stored in the refrigerator at -20° C until used.

Extracts and trappings of volatile substances of nests.

To obtain the attractive substances from nests, five nest shavings of lactating females (P7-8), free of feces, were used. The nest materials were placed in an Erlan Meyer (1 litre), heated to 60°C in a bain-marie. An air stream (250 ml/min) passed through the nest shavings and trapped the volatile odours in a clean glass tube in U form (3mm diameter, 16 cm long), cooled with ice + salt as refrigerant. After a 30 min trapping period, the glass U tubes were rinsed twice with 1 ml of polar solvent. The extracts were pooled and concentrated to 1 ml with nitrogen stream, stored in the refrigerator at -20° C until used for behavioural tests or GC-MS analysis.

Chemicals -All chemicals, including the 17 synthetic compounds as candidates for active constituents, were purchased from Sigma-Aldrich (St-Quentin Fallavier, France). They were >98% pure as determined by GC-MS analysis and were used without further purification. Each compound was diluted in polar solvent to obtain an adequate quantity for behavioural tests or for chemical identification with GC-MS. After pilot studies, we used 3 different doses: 500, 800 and 1,000 ng in 1µl dichloromethane unloaded on a filter paper for the Y maze test.

Chemical analysis -Gas chromatography-mass spectrometry (GC-MS).

The biological extracts and authentic compounds were analysed by gas chromatography-mass spectrometry (GC-MS), performed on a Thermo Finnigan PolarisQ Ion trap mass spectrometer with a Trace GC equipped with a split-splitless injector. An analytical capillary column Thermo Trace TR-5-MS was used (30m x 0.25 mm i.d., film thickness: 0.25 µm) with helium at 1 ml/mn as carrier gas.

Analyses were performed with a 1 µL splitless injection at 250°C, with an injection time of 1 min.

The temperature program was as follows: 1 min at 50 °C with 5 °C min-1 to 235 °C, held for 20 min and increased at a rate of 15 °C min-1 to 310 °C and finally held for 5 min. The transfer line and the manifold temperatures were at 280 and 200 °C, respectively. MS acquisition ranged from 50-650 in full scan mode. Compound identification was based on comparison of retention time and mass spectra of authentic standards as well as mass spectra comparison with the NIST (National Institute of Standard and Technology) mass spectral reference library.

Anethole exposure

To expose the foetuses and newborns to odorant during the gestation period and after parturition, we used anethole, a synthetic anisole compound. The novel flavour was incorporated in beverage water (0.5 µg anethole/1 ml water) and in chow food (1µg / 1gr chow) for three gravid and lactating females (anethole group). In addition, three gravid and lactating females were given food without anethole as control group. The nest shavings of lactating females (P7-8), free of feces, were used either for chemical extraction of active substances using trapping technique or for attraction stimulus in Y maze test.

Statistical analysis.

Differences in behavioural responses to stimuli were tested for statistical significance using Graphpad Prism6 software. The amount of time spent in direct contact nest was analysed by separate paired t-test (t-tests) in each group across individuals in a litter as statistical unit of analysis. All analyses were performed on raw performance data, not percentages. Fisher's test was used for analysis of performances in the direct contact nest and Y maze tests. P<0.05 was considered to indicate a significant difference.

Results

To identify the chemical composition of the MSO in rat, we developed consecutive stages to evaluate the attraction level between potential biological sources of active substances, to characterize the chemical compounds of these biological extracts, then to test the attraction power of identified molecules, and finally to investigate whether compounds in the diet can attract the pups (Fig. 1).

Attraction level between potential biological sources of active compounds

Two types of two choice behavioural tests were performed: the direct nest contact test and the Y maze test (Fig. 2A,B). In the direct nest contact test, postnatal day 5 (P5) pups were placed in the midline space between 2 types of nest shavings. The amount of time spent on the lactating female nest was higher than on clean or virgin female nests (Fig. 2C) (separate paired t-tests, P<0.05).

Significant preference for lactating female nest shavings was found in comparison to clean wood nests or virgin female nests, and no attraction difference was found between clean and virgin female nests (Fisher's, P<0.05)(Fig. 2D). These data indicate that the pups are attracted to the nest shavings of lactating females which are familiar to them and spend more time there. In addition, to refine this result, we performed Y maze tests wherein only the volatile substances emanating from substrates were submitted to the pups (P12-P14). The volatile substances of lactating female nests were more attractive for pups when they were opposed to clean, virgin female or male nest shavings (Fisher's, P<0.05)(Fig. 2E). The percentages of attraction responses for virgin nests in comparison with clean nest did not differ. We also tested the potential attraction of feces. No significant attraction of feces of lactating female was detected in comparison with that of male and of virgin female (Fisher's test, NS) (Fig. 2F). Only the lactating female nests contained attractive volatile substances that can be extracted in solvents for chemical analysis.

Characterization of the chemical compounds of biological extracts from gravid and lactating mothers

Volatile compounds of nest shavings of the lactating mother were extracted with polar (dichloromethane) and apolar (pentane) solvents. Newborns responded positively to mother's nest trappings extracted in polar and apolar solvents when paired with control solvents. As polar and apolar extracts elicit similar responses (Fig. 3A, Fisher's test, P<0.05), only the polar results were presented. To define the biological sources of these volatile attractive compounds, we investigated the effect of different tissue extracts from lactating female and from the AF of gravid female on P12-P14 pups. The volatile compounds of the nipple, ventral wash and milk from lactating females attract pups in the Y maze test in comparison with solvent as control (Fisher's, P<0.05) (Fig. 3B).

Pups also performed positive responses for nipples and crude AF of gravid rat early on day E18 of gestation, and for extracts of AF, when they were paired with solvent (Fisher's, P<0.05) (Fig. 3C). In our experiments, mother's ventral area, nipples, milk and pregnant AF were the sources of the MSO.

Then, gas chromatography-mass spectrometry (GC-MS) was used to identify specific or common molecules from ventral area, nipples, milk and AF extracts, together with extracts of nest and feces of lactating females, and rodent diet (Fig. 1 and4). We identified 37 compounds from these different extracts (Table 1). However, some compounds could not be tested or identified because of uninterpretable mass spectra caused by the lack of synthetic substances or by concentration or co-elution problems. Among the recognized molecules, 12 compounds were present in the AF and milk, 2 in the AF but not in milk, and 8 in the milk but not in AF. Four compounds were common to the diet and milk and only one to diet and AF (Table 1). Seventeen synthetic substances that could be potential MSO candidates were selected among the recognized compounds (Table 2). These compounds were used for identification of biological substances by comparison of their mass spectra. We showed that the spectrum of biological compounds found in tissue extracts was identical to that of synthetic compounds used for the Y maze (Fig. 5). Other molecules not tested in behavioural bioassays might be shown in the future to have a role in maternal signature odours.

Effect of characterized compounds in behavioural tests.

Only the behavioural tests in the Y maze allowed assessment of the biological properties of volatile active compounds. Among the seventeen molecules, six compounds, compared to solvent, attracted pups in the Y maze test and are considered as MSO constituents: diacetone alcohol, ethylbenzene, benzaldehyde, benzyl alcohol, methyl salicylate and vanillin (Table 1, Fig. 6A) (Fisher's, P<0.05). Gravid AF, nipples, and mother's nipples and milk contained 3 of these identified active substances in variable proportions: ethylbenzene (2-10%), benzaldehyde (3-10%) and benzyl alcohol (85-95%), presented with their mass spectrum (Fig. 3b). These 3 active compounds could be produced by gravid and mother rat itself, whereas diacetone alcohol and vanillin seemed to be acquired from the diet. These two compounds, diacetone alcohol and vanillin, are undetectable in AF by our systems of analysis, but may be present as well, although in trace quantities. Methyl salicylate was not detected in AF, and was not used in the blend (see below). These 6 attractive compounds are found associated with mother's nipple, ventral wash and nest trapping extracts.

Adult rat urinary volatile compound, 2-heptanone [START_REF] Osada | Profiles of volatiles in male rat urine: the effect of puberty on the female attraction[END_REF], was detected in nest trappings, but had no effect on pup attraction. Together, these findings suggest that the rat MAS was composed of ethylbenzene, benzaldehyde and benzyl alcohol, at least, present in AF, nipples and milk. Moreover, to attempt to define the natural MAS as a whole, we performed a synthetic MAS blend with the 3 common active compounds found in the AF, nipples and milk. The blend of these substances was prepared in similar proportions to those found in AF extracts, i.e. ethylbenzene, benzaldehyde, benzyl alcohol, in a ratio, respectively, of 1:1:12 (700 ng), according to chemical analysis. In behavioural tests, pups showed significant preference for the MAS blend in comparison with the control solvent (Fisher's, P<0.05) (Fig. 6B).

Attractiveness of an odorant incorporated in pregnant and lactating mother's diet

To attempt to determine the potential role of an odorant intake in MSO, we followed the route of an odorant, anethole, a synthetic anisole compound incorporated in pregnant and lactating mother's diet. Using GC-MS analysis, anethole was detected in AF, nipples, milk, and feces extracts of pregnant female and mother that consumed anethole. In Y maze tests, pups born from the mothers who consumed anethole were attracted by this aromatic compound (Fisher's, P<0.05) (Fig. 6C). In contrast, no attraction was detected for pups born from the mothers who did not consume anethole (Fig. 1 and6C). This experiment shows that previous experience of an odour is sufficient to elicit pup attraction.

Discussion

The first aim of this study was to examine the maternal attractive substances from rat lactating females that attract pups for suckling and try to provide new insights concerning the controversial origin of these attractive substances. The behavioural tests demonstrated that nest, nipples, milk, ventral area of lactating females, amniotic fluid and nipples of pregnant females are effective for attracting pups, but not feces. These data confirmed previous behavioural studies showing that AF, mother's ventral area, nipples and milk played a major role in nipple orientation, attachment and suckling process in newborn mammals [START_REF] Hofer | Evidence that maternal ventral skin substances promote suckling in infant rats[END_REF]Teicher andBlass, 1976, 1977;[START_REF] Singh | Oxytocin reinstates maternal olfactory cues for nipple orientation and attachment in rat pups[END_REF]Blass and Teicher, 1980;[START_REF] Rosenblatt | Olfaction mediated developmental transition in the altricial newborn of selected species of mammals[END_REF][START_REF] Varendi | Does the newborn baby find the nipple by smell?[END_REF][START_REF] Porter | Unique salience of maternal breast odors for newborn infants[END_REF][START_REF] Beauchamp | Early flavor learning and its impact on later feeding behavior[END_REF][START_REF] Schaal | Mammary odor cues and pheromones: mammalian infant-directed communication about maternal state, mammae, and milk[END_REF][START_REF] Schaal | Chemical signals 'selected for' newborns in mammals[END_REF]. AF is also an olfactory attraction source for newborns of several mammal species, including man [START_REF] Schaal | Responsiveness to the odour of amniotic fluid in the human neonate[END_REF][START_REF] Varendi | Attractiveness of amniotic fluid odor: evidence of prenatal olfactory learning?[END_REF][START_REF] Porter | Unique salience of maternal breast odors for newborn infants[END_REF][START_REF] Logan | Learned recognition of maternal signature odors mediates the first suckling episode in mice[END_REF].

The second aim was to identify, for the first time, the chemical composition of volatile MSO. MSO is a multi-component mixture, composed of 3 to 6 compounds depending on sources: 3 substances from amniotic fluid and nipples of pregnant females, 4 from ventral skin, 5 from milk and 6 from nipples, ventral wash and nest trapping of lactating females. These six substances have never been previously identified in rats as MSO components. The multi-component mixture of pheromone has already been underscored in many animal species (for review, [START_REF] Wyatt | Pheromones and animal behavior. Chemical signals and signatures[END_REF]. In AF, benzyl alcohol is the major compound with two minor substances, ethylbenzene and benzaldehyde. The level of positive behavioural performance reaction of the pups to the blend of these three compounds was very close to that for natural amniotic fluid. We did not found a synergy of the blend in the attractiveness performance as found for the nematode sex pheromone of Caenorhabditis elegans [START_REF] Pungaliya | A shortcut to identifying small molecule signals that regulate behavior and development in Caenorhabditis elegans[END_REF][START_REF] Forseth | NMR-spectroscopic analysis of mixtures: from structure to function[END_REF][START_REF] Lim | Common Carp Implanted with Prostaglandin F2α[END_REF][START_REF] Wyatt | Pheromones and animal behavior. Chemical signals and signatures[END_REF]. The positive behavioural performance level of the blend was similar to that for benzyl alcohol (500-800ng). However, this mixture could be suggested as a blend identity of rat species. These three individual compounds have been found as pheromone constituents in many animal species, especially insects (Pherobase) (www.pherobase.com). Moreover, benzaldehyde and benzyl alcohol were identified in relatively abundant amounts in human volatile metabolites of blood plasma collected from mother-infant pairs [START_REF] Stafford | Profiles of volatile metabolities in body fluids[END_REF] and in secretions of African wild dogs [START_REF] Wells | Prenatal olfactory learning in the domestic dog[END_REF]Apps et al., 2012), but the behavioural role of these compounds remains unknown. Likewise, benzaldehyde is contained in rabbit milk [START_REF] Schaal | Chemical and behavioural characterization of the rabbit mammary pheromone[END_REF] and in human milk [START_REF] Hausner | Characterization of the Volatile Composition and Variations between Infant Formulas and Mother's Milk[END_REF], without having a role in attraction of neonate. This compound has been identified as a pheromone component of more than 1 hundred insect species (Pherobase).

Ethylbenzene has been identified in bovine urine [START_REF] Kumar | Chemical characterization of bovine urine with special reference to oestrus[END_REF] and in Coffea arabica and used as a component of synthetic blend for coffee berry borer Hypothenemus hampei [START_REF] Mendesil | Semiochemicals used in host location by the coffee berry borer, Hypothenemus hampei[END_REF]. Furthermore, the 2-heptanone, dimethyl sulfone and benzaldehyde have been identified as urinary volatile compounds from mouse and rat [START_REF] Zhang | Putative chemical signals about sex, individuality, and genetic background in the preputial gland and urine of the house mouse (Mus musculus)[END_REF][START_REF] Zhang | Sex-and gonad-affecting scent compounds and 3 male pheromones in the rat[END_REF] and phenol, 4ethylphenol or indole [START_REF] Osada | Profiles of volatiles in male rat urine: the effect of puberty on the female attraction[END_REF] were also detected in our nest trapping, ventral wash or nipple extracts. However, these molecules were not attractive for rat pups. Dimethyl disulphide (DMDS), isolated from estrous hamster vaginal secretion, is found as an attractant for male hamsters (Singer et al. 1975). In rat, DMDS was shown to be effective in eliciting nipple grasping in 3-5 day-old pups, but with low releasing potency (about 50%) relative to that of olfactory intact nipples [START_REF] Pedersen | Olfactory control over suckling in albino rat[END_REF][START_REF] Schaal | Chemical signals 'selected for' newborns in mammals[END_REF]. DMDS is undetectable in rat bioactive extracts by our chemical system of analysis. While another sulfur compound, dimethyl sulfone, is identified in all bioactive extracts (table1) but is not attractive for newborn rats. Diacetone alcohol was identified in diet and detected in nipples, milk, nest trapping or ventral lavage. This hydroxyketone compound has been identified from anal gland of Tapinoma simrothi pheonicium ant as alarm pheromone [START_REF] Hefetz | Identification of new components from anal glands of Tapinoma simrothi pheonicium[END_REF]) and from temporal gland secretions of Alpine marmot Marmota marmota as a component of scent-marking substance (Bel et al., 1999). Vanillin, identified in nipples, ventral wash, nest trapping or faeces, and originating from diet, is utilised by many insect species in their communication system, and was also detected in human milk [START_REF] Buettner | A selective and sensitive approach to characterize odour-active and volatile constituents in small-scale human milk samples[END_REF]. Methyl salicylate, present at trace level in some rat extracts, is utilised by more than 80 insect species in their chemical communication systems (Pherobase). In addition, 2MB2, a volatile pheromone extracted from milk doe which facilitates suckling in young rabbit [START_REF] Schaal | Chemical and behavioural characterization of the rabbit mammary pheromone[END_REF], was not detected in rat MSO. On the same principle as for the first suckling episode in mice (Logan et al.2012), our data support the existence of 'maternal signature odours' rather than a classic pheromone that drives a hardwired response to specific or predetermined chemosensory cues. Our experiment with an exogenous molecule (anethole) incorporated in food is sufficient to enable pup behaviour (but not in no-anethole pups), which further reinforces this view.

The highlighting of a rat efficient volatile MSO, present in pregnant female's AF, and then in mother's nipples and milk, supports the hypothesis that the learning and memory mechanism processes would be activated in utero, as already suggested by [START_REF] Rosenblatt | Olfaction mediated developmental transition in the altricial newborn of selected species of mammals[END_REF], [START_REF] Porter | Unique salience of maternal breast odors for newborn infants[END_REF], [START_REF] Schaal | Mammary odor cues and pheromones: mammalian infant-directed communication about maternal state, mammae, and milk[END_REF] and [START_REF] Hepper | Long-term flavor recognition in humans with prenatal garlic experience[END_REF]. The relationship of biological rat MAS with intake of diverse diet odorants during the gestation period establishes the specific chemical signature of individual mother cues or 'maternal signature odours' (Logan et al.2012). This association opens new avenues for further investigating the neural mechanism underlying prenatal chemosensory learning and memory storage in the newborn brain receptors and behavioural specificity. Unravelling the chemical signals that cause innate attraction and searching for suckling, nipple attachment, foetal learning and memory in a mammal species is a necessary step in understanding the neuronal organisation pathways in signal reception and neuro-modulation in the formation of the new odorant receptors. Such a system may confer survival advantages by promoting the acquisition of information about safe foods. How does the plasticity process of the olfactory system operate in modifying its properties for specific environmental adaptation? Further dissection of the circuits engaged by this MSO should facilitate our understanding of the neurobiology of this evolutionarily conserved, innate social behaviour. financial interests or potential conflicts of interest. C, Anethole, the synthetic aniseed flavour, given in the diet of gravid and lactating rats, affected the chemical composition of amniotic fluid, milk, mother and gravid nipples. Only pups from 'anethole gravid rat' showed a chemosensory preference for anethole. In contrast, pups from 'no-anethole' gravid rat were not attracted by anethole. 1, listed in order of retention time. Behavioural active compounds were named on GC peaks. Peaks labelled "x" corresponded to the contaminated compounds such as complex siloxane or phthalate compounds. RT 17.70-Anethole indicated the retention time of synthetic anethole added to the diet of gravid female. Three identified compounds are common to extracts: Ethylbenzene, benzaldehyde and benzyl alcohol. Methyl salicylate was found in lactating female and nipples. Vanillin and diacetone alcohol, found in milk and nipples of lactating female, were derived from food. Anethole was found in all spectra when the female diet contained this substance. Vanillin and diacetone alcohol issued from food; 3 compounds, ethylbenzene, benzaldehyde, benzyl alcohol were found in the AF, milk and nipples; and methyl salicylate was present in milk and nipples but not in AF. (B) In Y maze, pups were attracted by a blend of the 3 synthetic compounds found in the AF (ethylbenzene, benzaldehyde, benzyl alcohol, in a ratio respectively of 1:1:12 for 700 ng in all). We suggest that this combination corresponds to natural MAS. (C), Attraction of pups to anethole that was introduced or not in the diet of rat female during pregnant and lactating period. Two experimental groups of pups were used: pups related to gravid females receiving anethole (A) and pups related to gravid females not receiving anethole (N). Only the "A" group pups were significantly attracted by anethole. This attraction suggests that food compounds, present in AF, contribute to the MSO and are involved in the prenatal learning and memory process.

Numbers indicate the numbers of pups tested. (*, P<0.05; Fisher's test) Bold and italic: attractive molecules found in AF or milk nipples or ventral wash but not in diet.

These substances potentially consisted of 'maternal signature odours'. Regular and Italic: attractive molecules found in diet. n: number of pups performing in Y maze test. Fisher's test, P<0.05 is significant.

Figure Captions Figure 1 /

 Captions1 Figure Captions

Figure 2 /

 2 Figure 2 / Behavioural effects on rat pups of different stimuli from nests and feces. A, schema of

Figure 3 /

 3 Figure 3 / Behavioural effects on rat pups of different stimuli from biofluids, and tissue extracts

Figure 4 /

 4 Figure 4 / Representative gas chromatography-mass spectrometry profiles of extracts.

Figure 5 /

 5 Figure 5 / Representative spectra of biological and synthetic compounds common to different

Figure 6 /

 6 Figure 6 / Behavioural effects of the identified compounds on pup attraction. (A) The 17

Table 1 :

 1 Compound identification by GC-MS from different extracts of gravid, lactating females and diet. The pick number indicated the identified compounds on GC profiles of different extracts in order of retention time (RT). MW: molecular weight. "x" corresponds to compounds identified in different extracts. Bold and italic: attractive compounds present in AF or milk or nipples or wash or nest but not in diet. Regular and italic: attractive molecules found in diet. *: compounds tested in behavioural tests. RT 17.70-Anethole indicates the retention time of synthetic anethole added to the diet of gravid female.Table 2: Statistical results for behavioural performance of compounds (500 and 1,000 ng) present in potential 'maternal signature odours'.

Acknowledgements. We thank V. Gilbert and E. Mansour for animal care, Ali Gharbi for assistance in the construction of the Y maze apparatus, B. Barascud for figures 2a,b, L. Pézard and N. Pech for statistics discussions, J. Gaudineau for providing some chemicals, M. Paul for correction of the English and four anonymous reviewers for their helpful comments.

Funding and Disclosure

The authors declare no conflict of interest. This work was supported by Aix-Marseille Université (AMU), the Centre National de la Recherche Scientifique (CNRS), The authors report no biomedical


Christophe Coudret

G Guirado

Cédric Hortholary

Jean-Pierre Launay

N Battaglini

Hubert Klein

P Dumas

"Electrical" Behaviour of Photochromic Compounds

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

Tel : 33(0)5 62 25 78 59, Fax : 33(0)5 62 25 79 99, email: coudret@cemes.fr One of the simplest molecular device of Molecular Electronics is a "bistable switch", a three terminal device for which a perturbation (input X) is used only to trigger a state exchange: the output R indicating only if state A or B is obtained . [ The two isomeric forms, interconverted by light-induced electrocyclic reactions, of photochromic dyes, are typical of a "bistable" compound. Furthermore, the large HOMO-LUMO gap variation accompanying the ring closure makes them attractive units for current switching. Known for their high stability, the photochromic dithienylethylene derivatives offer several advantages: high symmetry of both forms, and a polyene-type for the "ring-closed" one.

Numerous examples have been proposed as control unit for molecular devices,[2,3] and to our knowledge only one single molecule study have been performed, in broken junctions. [4] Scanning Tunneling Microscopy is a convenient way to address specifically this issue, but, since the molecule is then submitted to an electron flow, electrochemical properties of these compounds should also be taken in account. Indeed, recent reports indicate that ring-closure or -opening could be induced for the dithienylethene family by oxidation. [3,5,6] As a starting point we have selected very simple struct ures of the "molecular wire" type, or for electrochemical studies, bearing a trimethylene or a perfluorotrimethylene lateral cycle (figure 2). We wish here to present our preliminary result s in this domain.

Experimental

Synthetic details will be given elsewhere. Briefly, all compounds were obtained from the appropriate dihalo photochrome precursor. (scheme below)

S S X X R 6
Scheme (R = F, H ; X = Cl, I.)

Thus, 1 was prepared by a Sonogashira coupling from the known diiodide (R : F, X : I). [3] while compound 2 was obtained by lithiation of the described dichloride (R : H) [7] followed by trapping by diphenyldisulfide. Compound 3 was prepared following literature procedure. [7] Photochemical isomerisation was achieved using a TLC UV-lamp, and the resulting mixture of closed and open isomers was assayed by electrochemistry. Electrochemical behaviour was tested in oxidation by cyclic voltammetry (MeCN, 0,1M N(nBu) 4 PF 6 , glassy C, SCE , 1V/s) or by coulometry (plati num grid working electrode). Mixed SAM were prepared by exposure of gold substrate to solutions of the thioacetate 1 and n-octanethiol in dichloromethane; their surface was explored by STM in air, with very small current (<5 pA) in order not to destroy the SAM with the tip.

Self Assembled Monolayers

The highly oriented Mixed Self-Assembled Monolayers medium has been commonly proposed in the making of molecular devices. [8] Since it provides a « chemical like » environment to its components, fragile molecules can be studied as long as they dissolve in it, but, being only weakly conductive, they are difficult to image by STM, because it implies the detection of very low currents. Our first goal was to study the difference of conductance (electronic transparency) between two generated ex-situ photoisomers of a molecular wire containing the dithienylethene unit. By comparison to SAM of pure n-octanethiol, molecule 1 (open isomer) was found to be partly soluble in octanethiol, leading to demixed domains, and bright defects in the "octanethiol-rich areas" were attributed to the (more transparent than n-octanethiol) isolated molecules of 1.

More surprising was the fact that the contrast was found to be fluctuating at room temperature over minute time scale. In the present case, the reproducible blinking of the molecule was found to be bias voltage dependent. This fact was attributed to a chemical reaction. Although a current induced isomerisation is tempting, we investigated the possible oxidation of the molecule in these conditions since a simple change of electronic configuration may also lead to a different contrast.

Electrochemical behavior

As expected for both model compounds 2 and 3, the closed isomer is always easier to oxidize than the open one, however, the strongly electron withdrawing perfluorotrimethylene substituent shifts all potentials anodically. Thus, compound 3 's oxidation wave (irreversible) is centered at 1.99V vs. SCE : its oxidized ring-opened form is therefore a strong oxidizer.

Cyclic voltammetry analysis of compound 2 reveals that while the ring-closed isomer 2c shows reversible oxidation wave at 0.40V vs. SCE , the ring-open isomer 2o shows an irreversible oxidation wave at E pa =1.20 V respectively (figure 6).

Careful cyclic voltammetry analysis reveals that the irreversible oxidation wave of the ringopen isomer (ca. 1.20 V vs. SCE) on the first anodic scan, is followed by a reduction wave on the cathodic scan. The expected anodic counterpart is obtained on the second cycle giving a reversible wave centered on a redox potential value of 0.40 V vs. SCE. No specific substituent at the sigma bond locus are thus required to promote ring opening or closure (as opposed to ref .

[5a]), but it is found to depend strongly on the inductive effect of the saturated branch and also on the a substituent on the thiophene rings. Remarkably, this phenomenon occurs on very simple molecule and extension to larger one is underway.

Conclusion

Dithienylethene photochromes present a rich « electrical » behavior either in STM imaging or in electrochemistry. Rather easily oxidized, it is thus possible that their "ionized forms" may contribute to their electrical properties. Furthermore, depending on the subsituent, the ionized photochromic core can undergo analogous reaction to their photochemical one. Work is in progress to examine the possible correlation between the two phenomena, either by electrochemistry on larger compound, or by calculation.

"

  ELECTRICAL" BEHAVIOUR OF PHOTOCHROMIC COMPOUNDS C. Coudret, a G. Guirado, a C. Hortholary, a J.-P. Launay, a N. Battaglini, b H. Klein, b P. Dumas b A: Nanosciences Group, CEMES-CNRS UPR8011, BP94345,31055 TOULOUSE cedex 4 B: CRMCN Faculté des Sciences Luminy 163 av. de Luminy, 13288 MARSEILLE cedex 9

Figure 2 :

 2 Figure 2 : compounds studied in the present article

1 Figure 3 :

 13 Figure 3 : Schematic view of the proposed STM experiments

Figure 4 :Figure 5 :

 45 Figure 4 : Blinking of a photochrome in a n-octanethiol SAM (Scale 12.8x12.8nm, I : 3pA, Bias voltage 800 mV)

Figure 6 :

 6 Figure 6: a) CV of 2c (0.45 mM). b) CV of 2o (1.35 mM). Scan range: 1.00/0.00/+1.50/0.00

Acknowledgments

We gratefully acknowledge Prof. I. Gallardo, Universitat Autònoma de Barcelona, for electrochemical instrumentation, Mrs C. Viala for recording the NMR spectra. We also thank the "Ministerio de Educación, Cultura y Deporte" of Spain for the award of a postdoctoral grant (G.G.), the MENRST for fellowships (C.H., N.B.), and CNRS for a grant (AC-Nano, POEM)


Thomas Pottier
email: thomas.pottier@mines-albi.frwww.institut-clement-ader.org

Guénaël Germain

Madalina Calamaz

Anne Morel

D Coupard

Sub-Millimeter measurement of finite strains at cutting tool tip vicinity

Keywords:

published or not. The documents may come

Introduction

To achieve high performance machining, it is of high interest to collect data about the chip formation mechanisms. They can help to understand and improve the integrity of the machined surface, e.g. roughness, geometrical dimensions, microstructure, residual stresses, deformation levels, as well as the wear resistance of the tool [START_REF] Mabrouki | A contribution to a qualitative understanding of thermo-mechanical effects during chip formation in hard turning[END_REF]. To obtain quantitative data concerning transient strain, strain rate and temperature fields in very small areas (the primary Z I , secondary Z II and tertiary Z III deformation zones), numerical simulations have been developed which enable these parameters and their evolution to be evaluated. The results only have meaning if suitable friction laws [START_REF] Bahi | A new friction law for sticking and sliding contacts in machining[END_REF][START_REF] Childs | Friction modelling in metal cutting[END_REF] and material models [START_REF] Calamaz | Numerical simulation of titanium alloy dry machining with a strain softening constitutive law[END_REF][START_REF] Hor | An experimental investigation of the behaviour of steels over large temperature and strain rate ranges[END_REF][START_REF] Shi | Identification of material constitutive laws for machining -part i: An analytical model describing the stress, strain, strain rate, and temperature fields in the primary shear zone in orthogonal metal cutting[END_REF] are used. They are currently validated merely by macroscopic data, such as cutting forces, chip morphology and tool/chip contact length. Thus, details on the behavior in the cutting zone are still lacking due to the difficulty, through experimentation, for industrial cutting parameters, to obtain local data that may link with the simulations. Direct measurements of deformation field parameters, such as velocity of material flow and strain, have mostly been performed by quick-stop devices (QSDs). The fundamental idea is to retract the tool from the workpiece [START_REF] Jaspers | Material behaviour in metal cutting: Strains, strain rates and temperatures in chip formation[END_REF] or to accelerate the workpiece to separate it from the tool [START_REF] Buda | New methods in the study of plastic deformation in the cutting zone[END_REF] as rapidly as possible, in order to freeze the process. [START_REF] Komanduri | On the mechanics of chip segmentation in machining[END_REF] investigated the mechanics of chip segmentation in machining of a ferritopearlitic steel using an explosive QSD. They found that the chip segmentation process arises as a result of instabilities in the cutting process. [START_REF] Jaspers | Material behaviour in metal cutting: Strains, strain rates and temperatures in chip formation[END_REF] obtained clear photographs of chip roots on an AISI 1045 steel. Following the shear strain representation proposed by [START_REF] Ernst | Chip formation, friction and high quality machined surfaces[END_REF][START_REF] Lee | The theory of plasticity applied to a problem of machining[END_REF] they predicted strain in the primary shear zone. Another way to estimate strains is to mark the material with micro grids inscribed upon the side of the workpiece by mechanical abrasion [START_REF] Childs | A new visio-plasticity technique and a study of curly chip formation[END_REF], chemical abrasion or photo-etching lithographic printing methods as used by [START_REF] Pujana | In-process high-speed photography applied to orthogonal turning[END_REF]. The velocity and strain values are estimated from the micro grids distortion. [START_REF] Chaudhri | Subsurface deformation patterns around indentation in workhardened mild steel[END_REF] proposed a variation of the grid deformation technique, which could be used in machining, using steel specimens that contained pearlite 'stringers'. The stringers served as intrinsic internal grids whose distortion can be measured to estimate the strain. However a major drawback of QSDs is the time necessary to interrupt the cutting process. Even if improvements have been made in devices design [START_REF] Jaspers | Material behaviour in metal cutting: Strains, strain rates and temperatures in chip formation[END_REF], this delay time can trigger a modification of the deformation state in the cutting zone. Moreover experiments are very time-consuming since achieving a complete restitution of the formation sequence requires quick stops at various deformation stages.

An alternative to QSDs is the use of high-speed cameras. Indeed new performances in terms of acquisition frequencies, frame rate, exposure time, image resolution and signal-to-noise ratio, now allow to work close to industrial cutting speed. [START_REF] Pujana | In-process high-speed photography applied to orthogonal turning[END_REF] used such a technique on a square grid marked 42CrMo4 steel. They managed to analyze the deformed squares at a cutting speed up to 300 m/min, deduced strains and consequently strain rates in the primary shear zone on the hypothesis of plane strain conditions. Nevertheless, at such cutting speed, the capture apparatus is only able to render 1.5 images per segment and thus only provides a glance at a more complex process. More recently, [START_REF] List | Strain, strain rate and velocity fields determination at very high cutting speed[END_REF] proposed a method to replace the grids using flow line shape analysis. Four lines were drawn parallel to the cutting direction on the side of the workpiece using a mechanical scratching method. The velocity, strain rates and strain distributions were calculated from the mathematical expression of the streamline functions. Orthogonal cutting tests were conducted up to 1020 m/min. But a high feed of 0.84 mm had to be selected to guarantee a sufficient spatial resolution and avoid an underestimation of strain. The use of Digital Image Correlation (DIC) has also been reported to analyze the cutting zone. [START_REF] Hijazi | A novel ultra-high speed camera for digital image processing applications[END_REF] have developed a complex dedicated device composed of four non-intensified digital cameras set in dual frame mode to perform a 4 images acquisition at 1M Hz.

In the present paper, DIC technique has also been chosen. It is performed from the use of a single high frame rate imager and optical microscopy. Such experimental apparatus allows to monitor strain at several instants during the chip formation process. The following therefore presents first the experimental procedure. Details are given on cutting configuration and observation device. Then the DIC post-processing is described. Its accuracy is discussed. A following section is dedicated to the numerical estimation of strain from displacement fields. Finally the obtained results are presented and discussed. Mechanisms of chip formation are then proposed based on these experimental data.

Experimental setup

Material and cutting characteristics

The cutting configuration is chosen to be orthogonal. Cutting tests are performed on a planer machine GSP 2108 R.20 which allows varying the cutting velocity between 6 and 60 m/min. Uncoated carbide inserts with a tool holder manufactured for the planer machine (rake angle : 0 • ) are used. The tool is fixed and the cutting speed is applied to the machine table on which the workpiece is set up, along a direction perpendicular to the cutting edge (orthogonal cutting operation). The workpiece moves perpendicular to the observation axis.

The width of cut (w = 3mm) is given by the dimensions of the Ti6Al4V workpiece (100 × 60 × 3). A cutting depth of f = 0.25 mm is selected. This choice results from a balance between the need of large shear zone (lower magnification required) and the need to limit the out-of-plane effects (optical depth of field). The instruction of cutting speed is set to V c = 6 m/min. These cutting parameters are expected to generate serrated chips (see Fig. 1-c) on a material well known for segmenting even at low cutting speeds [START_REF] Komanduri | On the mechanics of chip segmentation in machining[END_REF][START_REF] Shaw | Machining titanium[END_REF].

Optics and field measurements

One of the main improvement of the present study is the use of high magnification optics allowing the capture of submillimeter zones of interest. For instance, in [START_REF] Gnanamanickam | Direct measurement of large-strain deformation fields by particle tracking[END_REF] the use of a 3X optical microscope allows a pixel size of 8.2µm at 250 f ps, in [START_REF] Guo | Deformation field in largestrain extrusion machining and implications for deformation processing[END_REF] the use of a high definition camera and a similar magnification allows a pixel size of 1.4µm at 2000 f ps. The work of [START_REF] Pujana | In-process high-speed photography applied to orthogonal turning[END_REF] proposes the use of a 12X video-microscope for a pixel size of 4.9µm at 25000 f ps. Finally, [START_REF] Hijazi | A novel ultra-high speed camera for digital image processing applications[END_REF] proposes to use 4 standard CCD sensors triggered successively in order to obtain 4 images with an inter-frame time of 0.001ms (equivalent to 1M f ps) at very high magnification (pixel size: 0.27µm). However, monitoring the chip formation process require a complete sequence of images. Therefore, the proposed capture process relies on a Photron Fastcam APX-RS camera.

The spatial resolution is a critical parameter in investigating large strain gradients, especially when localization phenomena are involved. The use of high speed camera limits quite drastically the number of measured pixels. Subsequently, the link between frame rate and frame resolution is responsible for limiting the maximum cutting speed of an observation. The choice of the capture parameters is governed by the will to obtain several images of one segment formation. In order to keep a sufficient spatial resolution, the acquisition is set at f = 18000 f ps with an exposure time of 6.6µs. This setting ensures a resolution of 384 × 352 pixels.

A tunable optical video-microscope is used and its magnification (M t ) is set to 35X. The field of view is then 650×600µm, with a pixel size of 1.65µm. With such settings, a cutting speed of V c = 6 m/min provides about 45 images of one segment formation (assuming that the width of a segment can roughly be approximated by f = 0.25 mm). One should also notice that such an optical apparatus is responsible for optical aberrations of unknown nature. Under this condition, one should be careful in the use of DIC when estimating the correlation between a reference (undeformed) image and a deformed one that has undergo serious displacement within the aberration field. Accordingly, iterative correlation should be preferred (see section ).

Lighting the scene is a non-trivial issue and deserve to be discussed further here. The presented images are captured using a 200W halogen light source and an optic-fiber light guide. Set at full power this device is not sufficient to capture images with an exposition time of 6.6µs. Actually the beam splitter embedded within the video-microscope is responsible for a significant loss of the incoming light flux (indeed the half of it). Therefore the light guide was positioned outside of the microscope tube, about 5mm away from the observed scene. The approximated angle of incidence is 30 • . Nevertheless, lighting remains one of the main constraint in high-speed imaging especially under microscopic conditions where over-exposure and/or under-exposure may become significant issues.

Digital Image Correlation Calculation parameters

The whole captured sequence is made of 2568 frames representing the formation of 52 chip segments. However, only one segment formation has been selected for post-processing purpose. Hence, 45 images are investigated. Each image is postprocessed through Digital Image Correlation (DIC) in order to retrieve the incremental displacement field. For this purpose, each image is split in square elements that create a virtual grid upon the sample surface (denoted M 0 = {x 0 ; y 0 }). The resolution of this grid (the gauge length) is set to 10 × 10 pixels, corresponding to 17 × 17µm 2 .The correlation process consists in looking for the most probable deformed pattern in the neighborhood of each node of this grid in terms of grey level. The displacement fields of each element are then assessed by the means of a bi-linear interpolation. Finally, the displacement at image n is assessed by correlating the n th image with the previous image (n -1) th . A zero-mean formulation of the correlation parameter is used in order to prevent from error due to non-constant lighting. The correlation parameter used in the present study are summarized in Table 1. Calculations are performed using 7D software [START_REF] Vacher | Bidimensional strain measurement using digital images[END_REF]. The choice of incremental correlation relies on several considerations. Firstly, as mentioned above, the optical aberration leads to a significant error when large displacements are involved ( [START_REF] Pottier | Study on the use of motion compensation techniques to determine heat sources. Application to large deformations on cracked rubber specimens[END_REF]) and comparing image n and n -1 instead of n and 1 decrease such displacements. Secondly, in presence of very significant strains, the likeness of image no.1 and n is poor and the correlation process cannot return information over the whole correlation grid M 0 . In other words, the material fed in the right hand side of image n has no counter part in image 1 for it is still out of the frame at that time.

Displacement uncertainties

Estimating the measurement uncertainties of speckle-related DIC techniques, is known to be a complex task. Lots of studies have been addressed in this field and the point of the present paper is not to discuss any further in this matter. However, the poor quality of the obtained images (low resolution, optical aberration, camera measurement noise, unpainted surface) give raise to the question of the accuracy/noise of the DIC obtained displacement fields. Among the several parameters impacting the DIC accuracy, speckle quality and pattern size are often addressed as the most critical ones [START_REF] Bornert | Assessment of digital image correlation measurement errors: Methodology and results[END_REF][START_REF] Pan | Study on subset size selection in digital image correlation for speckle patterns[END_REF].

The benchmark speckles generated numerically in [START_REF] Bornert | Assessment of digital image correlation measurement errors: Methodology and results[END_REF] offer a fast way to qualitatively investigate a given speckle. Indeed the estimation of the autocorrelation of the texture allows a comparison with the 3 proposed speckle. Fig. 2-a) show the texture and the autocorrelation surface of a 10 × 10 pattern. Fig. 2-b) depicts a comparison of the speckle under investigation with those proposed in [START_REF] Bornert | Assessment of digital image correlation measurement errors: Methodology and results[END_REF]. Hence, it can be seen that the obtained texture exhibits a sufficient quality to ensure a proper DIC (though between standard and coarse).

Another approach in evaluating speckle quality is the use of rigid body motion [START_REF] Triconnet | Parameter choice for optimized digital image correlation[END_REF]. A non-deformed zone of the image is investigated over 5 successive images and the displacement norm is monitored. The average displacement in the zone is then subtracted. Subsequently, the residual stands for the noise generated by speckle imperfection. Alike any random variable, it can be characterized by its standard deviation. Fig. 2-c) shows that for the chosen pattern size (10×10) the displacement uncertainties can be assumed to be about 0.0181 pixels ≈ 0.031µm (say a signal-to-noise ratio of 165 in the non-deformed zone).

Finally, the standard deviation of the measured displacement can also be estimated through the Mean Intensity Gradient (MIG) of the speckle pattern( [START_REF] Pan | Mean intensity gradient: An effective global parameter for quality assessment of the speckle patterns used in digital image correlation[END_REF]). It is shown that under some assumptions, the random error is given by:

std(u) ≈ √ 2σ n × δ f ( 1
)
where σ is the standard deviation of the measurement noise, N the subset size and δ f the MIG. In the present study the MIG equals δ f = 9.22 and the noise (σ = 1.025 pixel) is evaluated from the substraction of two consecutive images captured prior to any motion of the sample. Hence the standard deviation of the measured displacment is approximated by: std(u) ≈ 0.0157 pixels ≈ 0.027µm which is consistent with the rigid motion approach.

Out-of-plane motion

Unlike the work presented in [START_REF] Gnanamanickam | Direct measurement of large-strain deformation fields by particle tracking[END_REF], the cutting geometry does not impose constraint on the observed side of the chip. It means that the chip is free to swell along the optical axis of the microscope and therefore impose accounting for the out-of-plane deformation in the sheared section. The magnitude of the out-of-plane deflection is estimated from either SEM-MEX technique (stereoscopic 3D reconstruction Fig. 3a) and chromatic confocal surface metrology (Altimet 500). The approximated magnitude of the out-of-plane deflection is ∆z ≈ ±35µm for both measurements. However, one should notice that this measurement is performed post-mortem and does not account for the elastic deformation of the chip which is then neglected.

Several studies have been addressed on the estimation of DIC error due to out-of-plan motion and theoretical models enable the assessment of the errors raised from such motion. The work of [START_REF] Sutton | The effect of out-of-plane motion on 2d and 3d digital image correlation measurements[END_REF] proposes both a single lens model and a telecentric model to account for the virtual magnification of objects moving along the optical axis. The nature of the objective in used in this study (infitity corrected planapochromatic) disable the used of any of these models. Hence, the error estimation has been performed from experimental means. A planar speckled plate was moved along the optical axis and a pictured was captured every 10µm (within a range of -40µm to +40µm from the focus plane). DIC was then used to estimate the virtual strains due to out-of-plane motion. Fig. 3 shows that the error in terms of strains is maximized by ε max ≈ 0.0055. Since the swelling of the chip is not homogeneous over the chip segment, some regions are more affected by this error than others. However, the small magnitude of such error compared to the estimated strains (see section ) leads to consider it as negligible in the presented study case. One should notice that this error source can be minimized by the use of telecentric optics as disscussed in [START_REF] Sutton | The effect of out-of-plane motion on 2d and 3d digital image correlation measurements[END_REF] and [START_REF] Pan | High-accuracy 2d digital image correlation measurements with bilateral telecentric lenses: Error analysis and experimental verification[END_REF].

Strain Calculation

Finite strain Framework

The main difficulty in estimating displacement and strain fields relies on the use of incremental correlation which disable the tracking of a given material point. For every image, the increment of displacement is given at the undeformed/reference location as ∆U x (x 0 , y 0 , k)

∆U y (x 0 , y 0 , k) , (2)
where (x 0 , y 0 ) are the grid coordinates (identical for every image pair) and k is the image number. This only information does not enable the estimation strains (whether in initial or final configuration) since material tracking can not be achieved, cumulating strain and different location is just not right. The evaluation of the deformed coordinates {x k ; y k } at every step/images of the deformation process is performed through triangular bi-linear interpolation.

x k =x k-1 + ∆U x (x k-1 , y k-1 , k) y k =y k-1 + ∆U y (x k-1 , y k-1 , k) (3)
with

             ∆U x (x k-1 , y k-1 , k) = 3 i=1 φ i (x k-1 , y k-1 ) × ∆U x (x 0 , y 0 , k) ∆U y (x k-1 , y k-1 , k) = 3 i=1 φ i (x k-1 , y k-1 ) × ∆U y (x 0 , y 0 , k) (4
) where the φ i are the classical triangular shape functions. The knowledge of displacement increments offers a straight forward access to the velocity fields V x and V y . Assuming a constant capture rates of the images, it therefore comes V x = ∆U x and V y = ∆U y . Then the components of the strain rate tensor are:

                 D xx (x k , y k , k) = ∂V x (x k , y k , k) ∂x k D yy (x k , y k , k) = ∂V y (x k , y k , k) ∂x k D xy (x k , y k , k) = 1 2 ∂V y (x k , y k , k) ∂x k + ∂V x (x k , y k , k) ∂y k (5)
The cumulated displacement at material point location is then used to calculate the updated Lagrangian strain tensor as:

E (x k , y k , k) = D (x k , y k , k)+ k m=1 3 i=1 φ i (x k , y k )×D (x m , y m , m) (6)
where 1 < k < N f and N f is the number of frame in the sequence. E is then the cumulated strain with respect to the

Modal derivation

The major issue related to the implementation of Eq.( 6) comes from the noisy nature of displacements obtained by DIC approaches which lead to significant signal-to-noise ratio (SNR) when derived. Such numerical instabilities prevent from direct derivation of displacements when these latter exhibit high SNR, e.g.: natural speckle, small spatial resolution, inconsistent lighting, strain localization.. All of these problems arises with the combined use of high-speed photography and high magnification. Hence, filtering method should be considered and implemented.

In the present study, a modal projection method is used [START_REF] Pottier | A new filtering approach dedicated to heat sources computation from thermal field measurements[END_REF]. This method consist in projecting the displacement fields within a modal basis and build the strain fields from a weighted sum of modal space-derivative. The displacement increment is written as:

V x (x k , y k , k) = N p=1 λ p (k)Q p (x k , y k ) ( 7
)
where N is the truncation order (the number of modes used for reconstruction) here set to 350, λ p the modal coordinates and Q p the modal vectors. These latter are here evaluated from dynamics vibration theories. Indeed, prior to any calculation, the basis B = {Q 1 , Q 2 . . . Q N } is calculated by solving the classical dynamics problem:

M -1 K - 1 ω i I Q i = 0, ( 8
)
where M is the mass matrix, K the stiffness matrix and ω i the modal pulsation. The problem is solved using Finite Element discretization over a mesh build using one node per pixel of the captured image.

Once the velocity fields are projected within B the linearity of the derivative operator states that:

D ij (x k , y k , k) = ∂V i ∂x j = N p=1 λ p (k) ∂Q p (x k , y k ) ∂x j (9)
This approach, though uncommon in strain derivation applications, has proved its worth in various noise related problems [START_REF] Chrysochoos | An infrared image processing to analyse the calorific effects accompanying strain localisation[END_REF][START_REF] Wang | Shape features and finite element model updating from full-field strain data[END_REF][START_REF] Le Goic | Multi scale modal decomposition of primary form, waviness and roughness of surfaces[END_REF].

Results and discussion

Cumulated strain in a segment Fig. 4 shows the shape of the displacement and principal strain fields during chip segment formation. Adiabatic shear bands (ASB) are clearly highlighted by significant strain magnitudes (up to 3) at these locations. In addition, it can be noticed that the inner part of the segment does not significantly deforms (columns c) and d) image no.45).

Moreover, the actual cutting speed can be evaluated from the actual displacements increments measured in an undeformed zone of the image. For this purpose the displacement norm within the lower right side of the image (see Fig. 4-a) is averaged and gives V c ≈ 5.63 m/min which is slightly lower than the instruction (V c = 6 m/min).

As mentionned in the above, the measured strains are significant. However, a close monitoring of the image sequence shows the outbreak of a localized black line (Fig. 5-a). In order to determine the nature of this phenomenon, complementary SEM investigations have been carried out. As shown in Fig. 5b, a material disjunction (or crack ) appears on the free side of the chip.

The fact that, at some point of the deformation process, the material splits in segments leads to reconsider the strains magnitude assessment. The DIC process such as presented in this paper keeps cumulating strains even though material undergoes segmentation. Hence, the final strain magnitude within ASB is not relevant as such from a continuum mechanics standpoint. The values (up to 3) as depicted in Fig. 4-cd) are indeed greatly overestimated. It is then required to monitor the outbreak of this material disjunction.

For this purpose, the study of the strain rates within the ASB as depicted in Fig. 6 provides a straight forward way to monitor the crack propagation. From the curves presented in Fig. 6-b) it is noticeable that a segment is formed from three successive stages.

Stage 1 -The strains are concentrated at the tooltip vicinity. The inner part of the chip slowly deforms (Fig. 4-d). During this stage an out-of-plane swelling of the segment is clearly visible. It is fully formed around image no.30.

Stage 2 -The strain rapidly cumulates along the ASB from bottom to top. The strain rates reach significant magnitudes (up to 4.10 3 s -1 ) and lead to a material failure (this latter point is specifically discussed in section ) .

Stage 3 -During the last images, strain rates stabilize, the segment is fully formed and slips on both the cutting face and the next segment (to be formed). Indeed, since material failure occurs the segment is animated with a rigid body motion. At this point, cumulating strains is no longer valid from a continuum mechanics standpoint.

Fig. 6-b also shows that the times spent for stage 2 goes shorter when moving from point 1 to point 3. In other words, the material failure accelerates from the tooltip to the free surface. Considering the decreasing of the resisting cross-section, this results is indeed expectable. Fig. 7 shows the last measured state of strain before material failure (at the end of stage 2) for 19 points along the ASB. It can be seen that the deformation process that leads to failure slightly differs from one end of the ASB to the other. Different strain paths are observed and can be sorted in three categories:

Zone Z IA : Nearby the tooltip, the ASB deforms mainly in compression with significant magnitudes. This process is slow and start very early in the sequence (Fig. 6-b, point P 1 and Fig. 7-b).

Zone Z IB : Moving away from the tool tip, the ASB deforms in compression/shear but with lower magnitudes. The strain rates curves in Fig. 7-c show an acceleration of the phenomenon: the material is less deformed at failure in this zone but reaches it faster.

Zone Z IC : Finally, around the free surface, the state of strain turns into pure shear. At this time of the sequence the cutting speed reaches a cycle maximum. It is noticeable that the resisting cross section tends toward its minimum at the same time. It seems plausible that the two observations are related, however only a rigorous measurement of the cutting force may validate such hypothesis.

The study of strain rates enables to follow the crack tip when propagating along the ASB. This criterion, though unautomated yet, corroborates the visual observations of the images sequence. It allows to limit the strain accumulation and evaluate strains at failure. These latter appears to be heterogeneous and lower than expected by numerical calculation [START_REF] Shih | Finite element analysis of the rake angle effects in orthogonal metal cutting[END_REF][START_REF] Calamaz | Toward a better understanding of tool wear effect through a comparison between experiments and sph numerical modelling of machining hard materials[END_REF]. These observations points the need of a better understanding of the decohesion phenomenon and the segmentation process.

Chip segmentation process

The development of numerical models of machining operations has become significant in the last decade and the modeling of the segmentation phenomena is a key issue in the validation of material models (either for constitutive models and for damage evolution laws) [START_REF] Calamaz | Numerical simulation of titanium alloy dry machining with a strain softening constitutive law[END_REF]. A close monitoring of the image sequence reveals that the crack grows from the tooltip to the top free surface over 17 images. This film shot on the side surface, clearly shows that the disjunction goes from end to end. It can be seen in Fig. 8 that the material is disconnected on the side from δ = 10 µm while that disconnection appear at the center at approximately 70 µm. Say, a small part of the cross-section (in the triaxial pressure zone) is still attached to the next segment. It results in the formation of a serrated chip (and not a fully segmented one). As highlighted by [START_REF] Komanduri | New observations on the mechanisms of chip formation when machining titanium alloys[END_REF], the presence of a high hydrostatic stress zone at the chip center prevents form crack formation at the tooltip vicinity. As a matter of fact, such zone cannot exist near the free surface of the chip which is consistent with the end-to-end crack observed on the side (Fig. 5-a).

The formation process of this disjunction remains largely undocumented in the literature at least from an experimental standpoint and no mention of side effects is made. However, the observations performed during this work show significant differences between the side and core disjunction processes. From the knowledge of the 3D nature of the chip formation process and other published observations, one may think of three possible scenarios to model the crack evolution:

The first scenario is depicted in Fig. 9-a). The crack initiate at the tooltip (on the side), propagates along the primary shear band, generates a step on the top surface, and keeps propagating backward (through-thickness) to the triaxial pressure zone. This hypothesis is backed by chip post-mortem micrography and QSP observations of polished chips and is widely acknowledge in the scientific community [START_REF] Gentel | Chip formation in machining Ti6Al4V at extremely high cutting speeds[END_REF][START_REF] Vyas | Mechanics of saw-tooth chip formation in metal cutting[END_REF][START_REF] Nakayama | Machining characteristics of hard materials[END_REF]. Regretfully, these observations are made at one given time of the formation process and cannot provide evolutionary information. Moreover, the material removal due to polishing disable the observation of side effects.

The second scenario is depicted in Fig. 9-b). The crack initiates at the tooltip (on the side) and while propagating along the primary shear band it turns trough-thickness when the hydrostatic pressure becomes low enough. Then, it reaches the face-side corner first, then the center of the face. This scenario is consistent with the 2D numerical observations performed by [START_REF] Hua | Prediction of chip morphology and segmentation during the machining of titanium alloys[END_REF]. Authors have used the energy-based ductile fracture model proposed by [START_REF] Cockroft | Ductility and workability of metals[END_REF] and performed chip micrographic observations. However, the numerical model is built under a 2D assumption and does not provide information on the side effects.

The third scenario, depicted in Fig. 9-c), is actually a variation of the second scenario. Instead of a side initiation, the crack initiates all along the borders of the triaxial zone. One may assume that the high compressive strains at these locations make the triaxial zone behave as a rigid body that slips on the cutting face. The crack is then initiated simultaneously on the side and through-thickness. This scenario, though not as well-established as the others, may explain the existence of the material voids visible in various chip micrographic observations [START_REF] Courbon | Vers une modélisation physique de la coupe des aciers spéciaux : intégration du comportement métallurgique et des phénomènes tribologiques et thermiques aux interfaces[END_REF][START_REF] Braham-Bouchnak | Étude du comportement en sollicitation extrême et de l'usinabilité d'un nouvel alliage de titane aeronautique : le Ti555-3[END_REF].

Discussion among those three possibilities is a complex experimental (and numerical) task and the purpose of this work is not validate (or discard) these hypothesis. However, various observations can offer leads on that matter. For instance, the constant strain rate observed right after the completion of the side crack observed in Fig. 6 is not really compatible with the first and second scenarios (where the segment is still Step 1

Step 1

Step 2

Step 2

Step 3

Step 3

Hydrostatic stress zone c)

Step 1

Step 2

Step 3 not fully formed when the side crack runs from end to end). The first scenario does not explain the existence of material voids along the ASB. QSDs could be a good way to investigate the crack geometry but also existence and location of material voids and side effects, upon condition that the instant of the tool separation is precisely known (high frame rate imaging can be used on that purpose). In addition, the use of in-process observations of the top face may help differentiate the 3 scenarios (outbreak of the crack on the top). Another lead would be a cutting force monitoring. It would enable to link strain rates and the evolution of the resisting cross-section. Finally, the effect of temperature is also to be investigated.

Conclusion

This paper discusses the difficulties using built-in strain measurement in machining conditions. It proposes a submillimeter procedure for image acquisition and a suitable numerical post-processing that enables strain evaluation during the segmentation process. Results show a significant strain localization within the ASB and heterogeneity of the deformation behavior along this band. This work provides experimental evidences of a significant side effect and highlights the 3D nature of the chip (even in orthogonal cutting conditions). It therefore challenges the classical plane strain hypothesis widely acknowledged in literature. Indeed, chips are often wider than thick and this hypothesis holds as long as the averaged behavior is investigated. However, considering a chip formation process with crack initiation on the sides (where the plane stress hypothesis seems more suited) would clearly require the use of 3D finite element formulation.

In addition, the presented procedure offers a way to quantify the strains within the adiabatic shear band under the assumption of material continuity. However, as seen in the above, this hypothesis does not hold when serrated chip are produced. Hence, one understands that detecting the presence (or absence) of material disjunction is a serious issue and can lead to significant over-estimation of the strains. The development of a reliable criterion for crack detection (based on the orientation of the velocity gradient vector) is under construction and will be presented in future publication.

Two main drawbacks can be singled out of the presented experiments. The first one is the lack of in-process information on the magnitude and the shape of the out-of-plain deformation of the chip. It is a direct consequence of the chosen experimental procedure. Indeed, the topological study of this phenomenon is an experimental challenge of its own and constitutes a perceptive of this work. For this purpose, the use of stereo-imaging, computed tomography or any other non planar strain measurement technique seems inevitable.

Another drawback of the proposed procedure lies in the inability to measure strains at the direct vicinity of the cutting tool (i.e. in zones Z II and Z III ). Indeed, experimental constraints are responsible for the of data at tions. Firstly, the depth of field and the out-of-plane position of the cutting tool lead to a fuzzy delimitation of the tool edges (fuzzy parts of the image around the theoretical position of the cutting tool). Secondly, the nonalignment of the lighting with the optical axis leads to over-exposure of the disoriented facets, especially during the out-of-plane deformation of the segment (white regions of the chip). A correction of these experimental limitations should be addressed in future works.

Finally, achieving a complete comparability between machining experiments and models will require the investigation of thermal effects. Several attempts has been made [START_REF] Abukhshim | Heat generation and temperature prediction in metal cutting: A review and implications for high speed machining[END_REF][START_REF] Filice | On the FE codes capability for tool temperature calculation in machining processes[END_REF][START_REF] Arrazola | Analysis of the influence of tool type, coatings, and machinability on the thermal fields in orthogonal machining of AISI 4140 steels[END_REF], but the fulfillment of an energy balance over the formation of one segment remains unexplored and surely constitutes the most challenging perspective of this work.

Figure 1 :

 1 Figure 1: a) -Experimental apparatus [18]. b) -Geometry of the cutting and coordinates system. c) -Side view of the scene showing the camera capture frame and the serrated chip (SEM view overlaid).

Figure 2 :

 2 Figure 2: a) Image detail window 100 × 100. and autocorrelation zoom 10 × 10. b) Associated centered and normalized autocorrelation function radius at half height [4]. c) Distribution of normalized displacement vector norm.

Figure 3 :

 3 Figure 3: a) SEM image of the chip side, overlaid with the measured altitude map. b) Error estimation on strains due to out-of-plane motion.

17

 17

Figure 4 :Figure 5 :

 45 Figure 4: Displacements and principal strain fields at five time steps. The total elapsed time is 2.2 ms corresponding to the formation of one segment.a) Horizontal displacement (in µm). b) Vertical displacement (in µm). c) Major strain. d) Minor strain. The green rectangle represent the zone where displacements are averaged for the cutting speed estimation.

Figure 6 :

 6 Figure 6: a) Initial and final position of the three investigated points (P 1 , P 2 and P 3 ). b) Principal strain rates evolution over the 45 images of the film (at P 1 , P 2 and P 3 ).

Figure 7 :

 7 Figure 7: a) The three sub-zones of the ASB and the 19 selected points. b) State of strain at failure. c) strain rates at failure for the selected points.

Figure 8 :

 8 Figure 8: Successive cuts of the chip through thickness showing an open crack at the left end but connected material within the hydrostatic stress zone.

Figure 9 :

 9 Figure 9: a) First scenario : face-to-core propagation of the crack (with side initiation). b) Second scenario : side-to-core propagation of the crack (with side initiation). c) core/side-to-face propagation of the crack with simultaneous initiation.

Table 1 :

 1 Main parameters of the Digital Image Correlation

	Grid	pattern	grey level	displacement fields
	size	size	interpolation	interpolation
	10 × 10 10 × 10	bicubic	bilinear