Jessica Kojadinovic
email: jessica.kojadinovic@gmail.com

Christine H Jackson

Yves Cherel

George D Jackson

Paco Bustamante

Clos Saint

Multi-elemental concentrations in the tissues of the oceanic squid Todarodes filippovae from Tasmania and the southern Indian Ocean

Keywords: Cephalopod, Southern Indian Ocean, Tasmania, Metals, Bioaccumulation, Consumption guidelines

This study investigates 14 elements (Ag, As, Cd, Co, Cr, Cu, Fe, Hg, Mn, Ni, Pb, Se, V and Zn) in the tissues of the oceanic ommastrephid squid Todarodes filippovae from waters surrounding Ile Amsterdam (southern Indian Ocean) and Tasmania (Australia). As for other cephalopod species, the digestive gland and branchial hearts showed the highest concentrations of many elements (Ag, Cd, Se, V and Zn, and Cr and Ni respectively) highlighting their role in bioaccumulation and detoxification processes. With the exception of As and Hg, the muscles showed relatively low trace element concentrations. Squid size was positively correlated to Ag, As, Cd, Hg and Zn concentrations in Tasmanian squid and negatively correlated to all but Hg and Zn concentrations in Ile Amsterdam squid. Furthermore, no differences in elemental concentrations were noted between sexes. There were, however, some differences between mated and non-mated females from Tasmania. Comparing elemental concentrations in squid from both islands, higher concentrations of Cd, Co, Cr, Ni, Pb and V in squid sampled in Ile Amsterdam reflect different exposure conditions. When considering T. filippovae as a dietary resource for humans it should be noted that, given their Hg content, squids from Ile Amsterdam are not recommended for consumption on a regular basis. Moreover, regardless of the squid's origin, digestive glands should be avoided as Cd and Hg concentrations were above the European Union authorized limits in these organs.

Introduction

Trace elements in cephalopods have received increasing attention in recent decades, particularly in Europe and in Japan, as these molluscs play a major role both as predator and prey in marine ecosystems [START_REF] Boyle | Cephalopods: Ecology and Fisheries[END_REF], and are known to accumulate large quantities of Cd that can be transferred to higher trophic levels (e.g. Bustamante et al., 1998a;[START_REF] Dorneles | Cephalopods and cetaceans as indicators offshore bioavailability of cadmium off Central South Brazil Bight[END_REF].

Cephalopods also represent an increasing resource for humans [START_REF] Piatkowski | Impact of cephalopods in the food chain and their interaction with the environment and fisheries: an overview[END_REF].

Historically, the consumption of cephalopod products has been highest in countries of south-east Asia (e.g. Japan) and these countries still predominate in fisheries for cephalopods as well as in developing new technologies for catching them. In European waters, cephalopods have traditionally been a minor catch, although locally important in some southern European countries such as Portugal, Italy, Spain and Greece where cephalopods are high in local diets [START_REF] Payne | Trends and assessment of cephalopod fisheries[END_REF].

However their importance to European fisheries is increasing with the decline in traditional finfish stocks across the globe. For instance in the English Channel between 1983 and2003, total catches of cephalopods increased by almost 300%, from 8000 to 23000 tonnes [START_REF] Payne | Trends and assessment of cephalopod fisheries[END_REF].

Cephalopod fisheries are also becoming more important in other parts of the world, with the example of total cephalopod catches in Chile increasing by more than 5000% from 69 tonnes in 1978 to 3503 tonnes in 1996 [START_REF] Rocha | Overview of cephalopod fisheries in Chilean waters[END_REF]. This global fishery effort is likely to further increase in the next decade and species from remote areas could represent new fishery targets.

Besides their important ecological role, cephalopods display relatively short life spans, typically from one to two years in most of the targeted species. For this reason, they are of major interest for monitoring variations of pollutant concentrations in the environment as the accumulated concentrations in the cephalopod tissues reflect the bioavailability and metal variations in their immediate environment over a relatively short time scale [START_REF] Rossi | Mercury in Eledone cirrhosa from the Northern Tyrrhenian Sea: contents and relations with life cycle[END_REF][START_REF] Miramand | Variation of heavy metal concentrations (Ag, Cd, Co, Cu, Fe, Pb, V, and Zn) during the life cycle of the common cuttlefish Sepia officinalis[END_REF]. Indeed trace element analyses in marine organisms are commonly used to reflect the ambient concentrations, providing an integrated information over time according to the biological half life of elements in animals' tissues (e.g. [START_REF] Bustamante | Metal and metalloid concentrations in the giant squid Architeuthis dux from Iberian waters[END_REF][START_REF] Hédouin | Trends in concentrations of selected metalloid and metals in two bivalves from the SW lagoon of New Caledonia[END_REF]. This approach is particularly useful when trace elements are present at very low concentrations in the environment as it occurs in the Austral Ocean [START_REF] Honda | Distribution of heavy metals in Antarctic marine ecosystem[END_REF][START_REF] Fitzwater | Trace metal concentrations in the Ross Sea and their relationship with nutrients and phytoplankton growth[END_REF][START_REF] Sañudo-Wilhelmy | Trace metal distributions off the Antarctic Peninsula in the Weddell Sea[END_REF] and to compare different locations [START_REF] Rainbow | Biomonitoring of heavy metal availability in the marine environment[END_REF][START_REF] Warnau | Distribution of heavy metals in the echinoid Paracentrotus lividus (Lmk) from the Mediterranean Posidonia oceanica ecosystem: seasonal and geographical variations[END_REF].

Most of the studies concerning metal bioaccumulation have focused on species from the northern hemisphere, while very little data has been reported for cephalopods in the southern hemisphere. To the best of our knowledge, these studies are limited to selected metals such as Cd, Cu and Zn (e.g. Bustamante et al., 1998b;[START_REF] Dorneles | Cephalopods and cetaceans as indicators offshore bioavailability of cadmium off Central South Brazil Bight[END_REF]. In the Southern Ocean the importance of cephalopods in trophic webs is exacerbated as squid can occupy the ecological niche of epipelagic fish [START_REF] Rodhouse | Cephalopods occupy the ecological niche of epipelagic fish in the Antarctic Polar Front Zone[END_REF].

Although species of the genus Todarodes are particularly common in the northern part of this ocean, there is a paucity of biological information for the genus, particularly in relation to metal concentrations. Similarly there are surprisingly few data on metals for the well studied Japanese squid, Todarodes pacificus [START_REF] Tanaka | Subcellular distribution and binding of heavy metals in the untreated liver of the squid; comparison with data from the livers of cadmium and silverexposed rats[END_REF]Ichihashi et al., 2001b;[START_REF] Oikawa | Determination of trace elements in squid organs by inductively coupled plasma mass spectrometry and neutron activation analysis[END_REF][START_REF] Kim | Specific accumulation of heavy metals in squid collected from offshore Korean waters: preliminary results for offshore biomonitoring and food safety assessment[END_REF]. Records of metal concentrations in T. filippovae, which has a vast but remote distribution zone, are to our knowledge nonexistent. This paper describes the bioaccumulation and tissue distribution of 14 trace elements (Ag, As, Cd, Co, Cr, Cu, Fe, Hg, Mn, Ni, Pb, Se, V and Zn) in T. filippovae from waters neighboring the Subtropical Front 1 in the northern part of this species' distribution zone. These elements were selected as they represent, on one hand common essential elements accumulated by marine organisms, on the other hand non-essential elements the most likely to be hazardous to the squid's health and to that of its predators, including humans. The uniformity of elemental concentrations within this area is tested by comparing the results from two sampling zones located in the southern Indian Ocean and the southern Pacific Ocean (waters surrounding Ile Amsterdam and Tasmania, respectively) where T. filippovae appears to be the most abundant ommastrephid species [START_REF] Dunning | ) and[END_REF]. Furthermore, because T. filippovae is believed to have some fishery potential, the elemental concentrations obtained in this study are analyzed within the framework of metal concentrations associated with health risks to humans.

Materials and methods

Todarodes filippovae

Todarodes filippovae (Adam 1975) is a large epipelagic ommastrephid squid that can reach 50 cm (mantle length), although it commonly measures between 20 and 40 cm [START_REF] Rodhouse | Todarodes filippovae in the Southern Ocean: an appraisal for exploitation and management[END_REF]. It is characterized by a circum-polar distribution in the Southern Ocean from sub-Antarctic waters to slightly north of the Subtropical Front (south of 35°73 S), in association with high velocity currents [START_REF] Dunning | The Ommastrephid squid genus Todarodes: a review of systematics, distribution, and biology (Cephalopoda: Teuthoidea[END_REF][START_REF] Jackson | Temporal life history plasticity of the Southern Ocean squid Todarodes filippovae from waters off Tasmania, Australia[END_REF]. This oceanic species occurs mostly between the surface and 200 m in depth, in the open ocean up to the continental slope, and appears not to extend into shelf waters [START_REF] Rodhouse | Todarodes filippovae in the Southern Ocean: an appraisal for exploitation and management[END_REF]. Its life cycle is thought to be about a year. Spawning occurs in the Tasman Sea and off South Africa and probably takes place in the austral autumn and winter [START_REF] Rodhouse | Todarodes filippovae in the Southern Ocean: an appraisal for exploitation and management[END_REF][START_REF] Jackson | Temporal life history plasticity of the Southern Ocean squid Todarodes filippovae from waters off Tasmania, Australia[END_REF]. Todarodes species are opportunistic and will feed on fish as well as squid, or even have cannibalistic tendencies [START_REF] Boyle | Cephalopods: Ecology and Fisheries[END_REF]. Indeed, T.

1 Subtropical Front: A zone of enhanced meridional gradients of sea surface temperature and salinity in the poleward part of the subtropical convergence. A commonly used criterion that is found at the latitude at which the salinity, at a depth of 100 m, drops below 34.9 practical salinity units. In the Southern Hemisphere, the subtropical front can be traced from 40°S at the east coast of South America across the Atlantic into the Indian Ocean and across the Great Australian Bight, where it shifts to 45°S to pass south of Tasmania and reach the southern tip of New Zealand's South Island. It continues in the Pacific Ocean from the Chatham Rise east of New Zealand near filippovae feeds on myctophids and squid in Tasmanian waters [START_REF] Jackson | Temporal life history plasticity of the Southern Ocean squid Todarodes filippovae from waters off Tasmania, Australia[END_REF]. Todarodes species are likely to be an important part of the ecosystem in the Southern Ocean as a number of vertebrates prey on them including marine mammals (such as the sperm whale Physeter macrocephalus and the southern elephant seal Mirounga leonina), fish (e.g. the haddock Merluccius australis) and seabirds (e.g. albatrosses Diomedea melanophrys) [START_REF] Rodhouse | Todarodes filippovae in the Southern Ocean: an appraisal for exploitation and management[END_REF][START_REF] Cherel | Dietary evidence for spatial foraging segregation in sympatric albatrosses (Diomedea spp.) rearing chicks at îles Nuageuses, Kerguelen[END_REF][START_REF] Jackson | Temporal life history plasticity of the Southern Ocean squid Todarodes filippovae from waters off Tasmania, Australia[END_REF]. Although research on species identification, stock structure, and most aspects of its biology is still needed, T. filippovae is believed to have some fishery potential, with commercially viable catch rates having been reported in the Tasman Sea [START_REF] Rodhouse | Todarodes filippovae in the Southern Ocean: an appraisal for exploitation and management[END_REF]. First taken as bycatch to the Japanese jig fishery for Nototodarus sloani off New Zealand and southern Australia, it started being caught in commercial quantities off northeast Tasmania in 1978. In this region, it is the most abundant ommastrephid in the Subtropical Front and in slope waters off southeastern Australia [START_REF] Dunning | ) and[END_REF].

Sampling and preparation

T. filippovae were sampled from two locations, approximately 5 000 km apart, both situated slightly north of the Subtropical Front, in the northern part of the species distributional zone. The first location, in the Indian Ocean, lies roughly centered between the southern tip of Africa and Australia.

Twenty four individuals were collected from the research vessel La Curieuse during a cruise undertaken in April 2000. Squids were taken from 22 pelagic trawls (13 -380 m) during five consecutive nights in oceanic waters between 44°S, 76°E, and Saint Paul and Amsterdam islands (38°S, 78°105 E). Secondly, in the south Pacific Ocean, samples were obtained from waters around Tasmania (42°00'S, 147°107 00'E) which is situated off the south-east corner of the Australian mainland. Thirty two specimens were trawled in waters south-east of Tasmania in February/March 40°S and reaches the west coast of South America near 30°S (American Meteorology Society, 2010; [START_REF] Wikimedia | Wikipedia, l'encyclopédie libre[END_REF].

and July/August 2005. Samples were frozen in plastic bags, at -20°C, on board the sampling vessels.

Individual squid were defrosted and dorsal mantle length (ML) was measured to the nearest millimeter (mm). The total weight of each squid as well as the weight of the ovary or testis, digestive gland, gills and branchial hearts were weighed to the nearest gram (g). Significantly larger cephalopods were collected from Tasmanian waters (Table 1). Individuals were also sexed and females were examined as to whether they had recently mated. Subsamples of mantle muscle and digestive gland as well as the gills and branchial hearts were removed from all squid then freezedried, blended and ground to a fine powder to prepare for elemental determination. A subsample of the gonad was also removed from the Tasmanian squid and prepared in the same way.

Metal analysis

The metal analyses in samples from both study sites were realized simultaneously in the same batches of analyses. The total Hg concentrations in the powder obtained from the tissues were determined by analyzing Hg directly with an Advanced Mercury Analyzer (ALTEC AMA 254) on aliquots ranging from 5 to 50 mg of dry sample weighed to the nearest 0.01 mg (Bustamante et al., 2006b). The analysis of Ag, As, Cd, Co, Cr, Cu, Fe, Mn, Ni, Pb, Se, V and Zn required an extra step in the preparation protocol. From 20 to 300 mg of each sample were microwave digested in a mixture of 3 ml of suprapure nitric acid (VWR/Merck) and 1 ml of suprapure chloridric acid (VWR/Merck), and then diluted to 25 ml with deionized water. These 13 elements were then analyzed by Inductively Coupled Plasma Atomic Emission Spectrometry (Varian Vista-Pro ICP-AES) and Mass Spectrometry (ICP-MS II Series Thermo Fisher Scientific) in all tissues but the gonads. To avoid metal contamination, all glass and plastic utensils used were washed with detergent, soaked in a bath of mixed nitric (35 ml.l -1 ) and chlorhydric (50 ml.l -1 ) acids for a minimum of 24 h, rinsed 3 times in deionized (Milli-Q quality) water and dried in an oven at 50°C before use.

Accuracy and reproducibility of the preparation were tested by preparing an analytical blanks and replicates of lobster hepatopancreas (TORT-2) and dog-fish liver (DOLT-3) reference standards (National Research Council, Canada) along with each set of samples. Recovery rates were respectively equal to 91 ± 9% for Ag, 95 ± 23% for As, 92 ± 7% for Cd, 83 ± 8% for Co, 105 ± 40% for Cr, 94 ± 8% for Cu, 87 ± 8% for Fe, 100 ± 3% for Hg, 96 ± 14% for Mn, 95 ± 15% for Ni, 109 ± 23% for Pb, 154 ± 23% for Se, 96 ± 7% for V, and 101 ± 12% for Zn. Element concentrations are expressed in µg.g -1 of dry weight (dw).

Data analysis

Besides classical numerical summaries and plots, the 149 following statistical analyses were performed at the 95% confidence level while carefully observing the p-value in the analysis. First, ttests were used to assess whether the squid sampled in Tasmania were significantly different from those sampled in Amsterdam in terms of the biological measurements (Table 1). Second, one-way ANOVAs for paired samples were used to assess whether there were significant differences between concentrations of the same metal in up to five tissues. In addition, in order to test the significance of differences in As and Cu concentrations between gills and the others tissues of Tasmanian squid, we performed paired t-tests and adjusted their p-values using Bonferroni's correction (which is known to be a conservative method). Third, ANCOVAs, using the log-transformed mantle length as covariate, were used to test the effect of sex (in Amsterdam squid), of the mated status (in Tasmania squid) and of the geographic site on the log-transformed elemental concentrations. This section documents the concentrations of trace elements in the tissues of T. filippovae caught in Ile Amsterdam and Tasmanian waters (see Tables 2 and3, andFigures 1, 2 and3), as well as describes factors that were shown to influence trace element concentrations in this species.

However, for some elements, concentrations were below the detection limits. In such cases ("< DL" symbol in Tables 2 and3) the results were not included in any further analyzes.

Organotropism

The digestive gland contained the highest concentrations of Ag, Cd and Zn of all the tissues analyzed, irrespective of geographic location. Similarly, the highest concentrations of Co and Cu as well as Fe were found in the digestive gland of squid form Ile Amsterdam and Tasmania respectively (Figures 1, 2 and 3). The branchial hearts had the highest concentrations of Cr and Ni in both locations along with the highest concentrations of Hg and Fe in Ile Amsterdam squid and Co in Tasmanian squid. However, the gills were the major accumulation tissues for As, Cu and Mn in T. filippovae caught around Tasmania (for As p<0.01 between gills and digestive gland and p<0.001 between gills and muscle; for Cu p<0.001 between gills each other tissues: branchial hearts, digestive gland and muscle; for Mn p<0.01 between gills and muscle). On the other hand, the highest concentrations of Hg in Tasmanian squid were found in the muscle. For the three tissues in which there were detectible concentrations of As, the highest amounts were found in the gills of the Tasmanian squid and in the muscle of the Ile Amsterdam squid. Furthermore, detectible concentrations of Pb were highest in the gills of Tasmanian squid but for Ile Amsterdam squid, the digestive gland had the highest concentration of Pb. Levels of Se and V were highest in the digestive glands from both of the study areas. Overall, the digestive gland stands out as the tissue of greatest importance in terms of elemental accumulation followed by the branchial hearts, the gills and the muscle.

Squid biological variables

Among the measurements taken on each individual (viz. mantle length, total weight, ovary weight, digestive gland weight, gills weight and branchial heart weight), mantle length was found to be the most correlated to the concentrations of trace elements (Table 4). Moreover mantle length was very well correlated to the weight measurements (data not shown) and was thus chosen as a covariate when testing (by means of ANCOVAs) the effect of sex, mated status and geographic site on elemental concentrations.

The influence of gender was tested only in Ile Amsterdam as there were no males among the squid collected in Tasmania. [START_REF] Jackson | Temporal life history plasticity of the Southern Ocean squid Todarodes filippovae from waters off Tasmania, Australia[END_REF] have noted that males seemed better at avoiding the trawl nets than females. No significant difference in elemental concentrations was detected between male and female squid of equal mantle length (Table 5). Mated females were found only from Tasmanian waters. The comparison of elemental concentrations between mated and non-mated females of similar sizes showed no significant differences in the branchial hearts, gills or gonads. However, some weak evidence was found for As and Cu in the muscle, and for Cr and Mn in the digestive glands (Table 5). There were lower concentrations of Cu and higher concentrations of As, Cr and Mn in mated females (data not shown).

Geography

The size-normalized comparison of the squid sampled in Ile Amsterdam and in Tasmania indicated significant differences in concentrations of all elements but Mn and Se, although not for every element/tissue combination (Table 5). Levels were higher in Ile Amsterdam than in Tasmania for Cd, Cr, Ni and Pb in all tissues, and equal or higher for Co and V according to the tissue (Tables 2 and3 andFigures 1, 2 and3). On the contrary, concentrations were higher in Tasmania for As in all tissues, and equal or higher for Cu, Hg and Zn according to the tissue. The results for Ag and Fe were opposite in different tissues. For Ag, concentrations in the digestive gland were highest in squid from Ile Amsterdam whereas, in the gills they were highest in squid from Tasmania. For Fe, concentrations in the branchial hearts were highest in squid from Ile Amsterdam whereas, in the digestive gland, they were highest in squid from Tasmania. Overall, particularly large differences in concentrations between both sites were observed for Cd, Co, Cr, Cu in the gills, Fe in the branchial hearts and the digestive gland, for Hg in the muscle tissue and for Ni in the branchial hearts (Figures 1, 2 and3).

Discussion

Trace element concentrations in cephalopods have received increasing attention over the last decades as these molluscs play a major role both as predators and prey in marine ecosystems, and are widely consumed by man (e.g. [START_REF] Boyle | Cephalopods: Ecology and Fisheries[END_REF][START_REF] Pierce | Geographic, seasonal and ontogenetic variation in cadmium and mercury concentrations in squid (Cephalopoda: Teuthoidea) from UK waters. Ecotox[END_REF]. Previous studies have demonstrated their ability to rapidly accumulate high concentrations of trace elements in their tissues making them good bioindicators of environmental levels but also a potential source of human intoxication (e.g. [START_REF] Storelli | Cadmium and mercury in cephalopod molluscs: Estimated weekly intake[END_REF][START_REF] Storelli | Intake of essential and non-essential elements from consumption of octopus, cuttlefish and squid[END_REF][START_REF] Pernice | Comparative bioaccumulation of trace elements between Nautilus pompilius and N. macromphalus (Cephalopoda: Nautiloidea) from Vanuatu and New-Caledonia[END_REF]. As in most marine animals, concentrations may vary with biological factors specific to an individual, a population or a species, or with environmental factors [START_REF] Pierce | Geographic, seasonal and ontogenetic variation in cadmium and mercury concentrations in squid (Cephalopoda: Teuthoidea) from UK waters. Ecotox[END_REF]. The impact of such factors on the elemental concentrations found in Todarodes filippovae are discussed hereafter in the aim to facilitate the future use of this species as a bioindicator of environmental element levels. Finally, the results are regarded from a human health perspective.

Metal distribution in the tissues

Many studies highlight that in cephalopods, the digestive gland has a central role in the bioaccumulation of trace elements, particularly for toxic metals such as Ag and Cd (see e.g. [START_REF] Martin | High copper concentrations in squid livers in association with elevated levels of silver, cadmium, and zinc[END_REF]Ichihashi et al., 2001a;Bustamante et al., 2002bBustamante et al., , 2004a;;[START_REF] Miramand | Variation of heavy metal concentrations (Ag, Cd, Co, Cu, Fe, Pb, V, and Zn) during the life cycle of the common cuttlefish Sepia officinalis[END_REF].

The branchial hearts are considered as excretion tissues, allowing the depuration and/or the storage of various elements (e.g. [START_REF] Nardi | Isolation and distribution of adenochrome(s) in Octopus vulgaris[END_REF][START_REF] Guary | High concentration of transuranic and natural radioactive elements in the branchial hearts of the cephalopods Octopus vulgaris[END_REF]Bustamante et al., 2002b).

Consequently, in most cephalopod species, these two tissues contain the highest concentrations of many trace elements. This was also the case in this study of T. filippovae, irrespective of location, particularly in relation to Ag, Cd, Se, V and Zn, and Cr and Ni that were concentrated in the digestive gland and the branchial hearts respectively. It is interesting to note that, in many marine animals, Ag, Cd, Cu, Hg and Zn bind to metallothioneins as detoxification and homeostasis processes [START_REF] Anan | Subcellular distribution of trace elements in the liver of sea turtles[END_REF]Bustamante et al., 2004a). Although these phenomena are not well documented in cephalopods [START_REF] Tanaka | Subcellular distribution and binding of heavy metals in the untreated liver of the squid; comparison with data from the livers of cadmium and silverexposed rats[END_REF][START_REF] Craig | Metal in squid, Loligo forbesi, adults, eggs and hatchlings. No evidence for a role for Cu-or Zn-metallothionein[END_REF]Bustamante et al., 2006a), the joint concentration of Ag, Cd, Cu and Zn in the digestive gland of T. filippovae may well be an indication that detoxification mechanisms exist to hinder the toxic action of both essential and non-essential metals in this species. The strongest metal concentration correlations were observed in the digestive gland between Ag, Cd, Cu and Zn in squid from Amsterdam (correlation coefficients ranging from 0.69 to 0.89, with 266 p-values <0.001), and between Ag and Cu in squid from Tasmania (r=0.79, p<0.001). The correlations cited above may be the sign of the joint binding of these metals to metallothioneins.

Another alternative route for metal elimination in female squid is the transfer of metals to their eggs, as this has been observed in experimental conditions for Ag, Se and Zn [START_REF] Lacoue-Labarthe | First experiments on the maternal transfer of metals in the cuttlefish Sepia officinalis[END_REF]. No significant differences in Cd and Hg concentrations between mated and non-mated females were observed in this study suggesting that these two elements are not likely to be excreted through the eggs in T. filippovae. The transfer to the eggs might however be specific to certain elements such as essential ones [START_REF] Lacoue-Labarthe | First experiments on the maternal transfer of metals in the cuttlefish Sepia officinalis[END_REF]. Levels of Cu in muscle were found to be significantly lower in mated individuals than in non-mated ones suggesting a transfer of Cu from the muscle to the gonads in the mated females as this element is essential for the development of the hatchlings [START_REF] Villanueva | Composition in essential and non essential elements of early stages of cephalopods and dietary effects on the elemental profiles of Octopus vulgaris paralarvae[END_REF].

In individuals originating from Tasmania, the gills concentrated most of the As, Cu and Mn. The majority of the elements present in cephalopods are assumed to be incorporated by the diet as these carnivorous animals have a high feeding rate (Bustamante et al., 2002b;[START_REF] Villanueva | Composition in essential and non essential elements of early stages of cephalopods and dietary effects on the elemental profiles of Octopus vulgaris paralarvae[END_REF]Bustamante et al., 2006c). However direct uptake of elements from seawater through the gills is also a known phenomenon in cephalopods (Bustamante et al., 2004b(Bustamante et al., , 2006c)). It has been verified in different species for elements such as Ag, Cd and Zn [START_REF] Ueda | Accumulation of 65 Zn by octopus Octopus vulgaris[END_REF][START_REF] Koyama | Bioaccumulation of waterborne and dietary cadmium by Oval Squid, Sepioteuthis lessoniana, and its distribution among organs[END_REF]Bustamante et al., 2002aBustamante et al., , 2004b) ) and suggested for elements such as Cr, Fe and Ni (Miramand et al., 2006;[START_REF] Bustamante | Metal and metalloid concentrations in the giant squid Architeuthis dux from Iberian waters[END_REF]). It appears from the present results that seawater may also be a direct source of As and Mn in some cephalopods under certain conditions (in Ile Amsterdam the gills were not the target tissue for As). It would be interesting to further pursue this hypothesis as, to the best of our knowledge, there exist no experimental studies on the absorption of these elements via the gills. The results for Cu were quite striking, with concentrations, two, three and 58 fold higher in the gills than in the branchial hearts, the digestive glands and the muscles, respectively.

Copper is however usually present in low concentrations in seawater [START_REF] Watanabe | Trace minerals in fish nutrition[END_REF]. The most straightforward explanation for such a gill-oriented accumulation of this essential element is its association with haemocyanin. Indeed, Cu works as a respiratory pigment in haemocyanin, which is the typical dioxygen carrier for molluscs and crustaceans that represents 98% of blood protein in cephalopods [START_REF] Ghiretti | Molluscan hemocyanins[END_REF][START_REF] D'aniello | Electrolytes and nitrogen compounds of body fluids and tissues of Octopus vulgaris Lam[END_REF]. Due to the respiratory function of the gills, it would appear highly likely to observe a greater presence of haemocyanin and hence Cu in the gills.

The muscle tissue of T. filippovae was characterized by the lowest concentrations of most trace elements. This result is consistent with data reported for other cephalopod species [START_REF] Miramand | Concentration and distribution of heavy metals in tissues of two cephalopods, Eledone cirrhosa and Sepia offcinalis, from the French coast of the English Channel[END_REF][START_REF] Storelli | Cadmium and total mercury in some cephalopods from the South Adriatic Sea (Italy)[END_REF]Ichihashi et al., 2001b;[START_REF] Bustamante | Metal and metalloid concentrations in the giant squid Architeuthis dux from Iberian waters[END_REF]. There were two exceptions, for As and Hg, which were accumulated mostly in the muscle of cephalopods from ^Ile Amsterdam and Tasmania, respectively. Arsenic has been shown to have a tropism towards muscular tissues in cephalopods [START_REF] Pernice | Comparative bioaccumulation of trace elements between Nautilus pompilius and N. macromphalus (Cephalopoda: Nautiloidea) from Vanuatu and New-Caledonia[END_REF], but few data exist on its speciation. The As accumulated in the muscle tissue of T. filippovae could be mostly present in its organic form, hence non toxic, but further research would need to be conducted to corroborate this possibility. A Hg organotropism towards muscle has also previously been described by various authors including (Bustamante et al., 2006b), who related it to the existence of a Hg excretion function of the digestive gland and/or a preferential redistribution of Hg (mostly in the organic form) to muscular tissues where it binds to sulphydryl groups of proteins. Such chemical transformations are also well described in fish in which virtually 100% of the Hg is in the methylated form [START_REF] Bloom | A survey of size-specific mercury concentrations in game fish from Maryland fresh and estuarine waters[END_REF]. Since food appears as the main pathway of exposure for Hg (Lacoue-Labarthe et al., 2009b) and T. filippovae mainly feeds on mesopelagic fish in both areas (Jackson et al., 2007, Cherel unpublished data), the reasons for such a difference in the accumulation processes between T. filippovae from Tasmania (Hg mostly accumulated in the muscle) and those from Ile Amsterdam (Hg mostly accumulated in the branchial hearts) appear unclear.

Influence of biological variables

The potential impact of certain biological factors are considered here, such as body and organ measurements, sex and whether or not the individual had mated that year.

Size-related elemental concentrations

The most obvious result was the correlation between the size (viz. mantle length) of T. filippovae and the trace element concentrations found in its tissues (Table 4). Such an observation is common across a large spectrum of marine species. Most often, given the bioaccumulating properties of trace elements, their concentrations tend to increase with the size (and presumably age) of the individual. This was the case for Ag, As, Cd, Hg and Zn in Tasmanian squid and for Hg and Zn in Ile Amsterdam squid. On the contrary, for all the other elements tested in squid from Ile Amsterdam, concentrations decreased with squid size. This denotes a greater tendency of these elements to accumulate in small sized animals rather than in larger ones. A plausible explanation for the negative correlations between these metals and squid size could be attributed to the marked modification in T. filippovae's diet during its life cycle. It is common for squid to experience a drastic change in diet during ontogeny, young squid consuming mainly crustaceans and older squid living primarily on fish [START_REF] Pierce | Geographic, seasonal and ontogenetic variation in cadmium and mercury concentrations in squid (Cephalopoda: Teuthoidea) from UK waters. Ecotox[END_REF]. This hypothesis concurs with recent findings stemming from isotopic studies on the very same animals used in this study, indicating the existence of dietary shifts from lower to higher trophic levels during growth [START_REF] Cherel | Tissue, ontogenic and sex-related differences in δ13C and δ15N values of the oceanic squid Todarodes filippovae (Cephalopoda: Ommastrephidae)[END_REF]. Another explanation could be the existence of a "dilution" effect linked to the rapid increase of tissue mass due to growth (that is very rapid in cephalopods) with regards to proportionally lower intake of trace elements [START_REF] Pierce | Geographic, seasonal and ontogenetic variation in cadmium and mercury concentrations in squid (Cephalopoda: Teuthoidea) from UK waters. Ecotox[END_REF].

Sex and mated status

Contrary to expectations, no differences in metal concentrations were noted between sexes in spite of differential growth rates enabling females to reach greater mantle lengths and weights than males [START_REF] Jackson | Temporal life history plasticity of the Southern Ocean squid Todarodes filippovae from waters off Tasmania, Australia[END_REF][START_REF] Cherel | Tissue, ontogenic and sex-related differences in δ13C and δ15N values of the oceanic squid Todarodes filippovae (Cephalopoda: Ommastrephidae)[END_REF]. This suggests, however, that both sexes share the same physiology regarding uptake and depuration of trace elements, as previously suggested during exposure to radioisotopes of a range of metals in Sepia officinalis (Bustamante et al., 2002b(Bustamante et al., , 2004b(Bustamante et al., , 2006c;;Lacoue-Labarthe et al., 2009a). Additionally, this also suggests that males and females of similar sizes have a similar diet as the trophic pathway is the most important one for the majority of trace elements (Bustamante et al., 2002a;Lacoue-Labarthe et al., 2009a). Among females however, differences in muscular Cu concentrations and concentrations of Cr and Mn in the digestive gland, seem linked to the reproductive state (Table 5). Some of these differences could be due to a redistribution of these essential elements within the organism of the mated individuals.

Influence of geography

Among environmental factors, the geographical origin often has a considerable impact on trace element concentrations in cephalopods. The environment dictates the quantity of trace elements available, either directly by water absorption or indirectly through the ingestion of impregnated prey items.

For a recent review of metal concentrations in different cephalopod species from various origins refer to [START_REF] Bustamante | Metal and metalloid concentrations in the giant squid Architeuthis dux from Iberian waters[END_REF], Table 3. Overall, the metal concentrations measured in the digestive gland of T. filippovae during this study are within the spectrum of values found in the digestive gland of cephalopods worldwide, often towards the lower end, specially for Pb and V.

With the exception of Cd, the concentrations of non-essential elements resemble particularly those measured in the closest related species, T. pacificus and Stenoteuthis oualaniensis, sampled off Japan (western Pacific Ocean). Cadmium concentrations in Ile Amsterdam were higher than in most other cephalopods and similar to another ommastrephid species, Ommastrephes bartrami, sampled off the California coast (eastern Pacific Ocean). It is noteworthy that the concentrations of Cr and Ni reported here are among a very small body of information on these metals in cephalopods. In comparison, Ni and Cr concentrations in the digestive gland of T. filippovae were 3 to 30, respectively 5 to 31, times lower than concentrations measured in nautilus species from New Caledonia and the Vanuatu archipelago [START_REF] Bustamante | Bioaccumulation of 12 trace elements in the tissues of the nautilus Nautilus macromphalus from New-Caledonia[END_REF][START_REF] Pernice | Comparative bioaccumulation of trace elements between Nautilus pompilius and N. macromphalus (Cephalopoda: Nautiloidea) from Vanuatu and New-Caledonia[END_REF]. However, the concentrations of essential elements seem to be more variable among species in relation to the specific physiological needs of each animal rather than purely dependent on the environment. For instance, [START_REF] Pernice | Comparative bioaccumulation of trace elements between Nautilus pompilius and N. macromphalus (Cephalopoda: Nautiloidea) from Vanuatu and New-Caledonia[END_REF] showed that nautilus living in an environment enriched in Ni (from Ni mining in New Caledonia) did not have higher Ni concentrations than a closely related nautilus species living in an environment not Ni-enriched (Vanuatu archipelago).

In this study, T. filippovae showed significantly different elemental concentrations according to their location. Higher concentrations of Cd, Cr, Ni and Pb (and to a lesser extent Co and V) in squid sampled in Ile Amsterdam compared to those caught in Tasmanian waters could be an indication that these elements are present in higher concentrations and/or more available in the Ile Amsterdam environment. On the contrary, concentrations of As (and to a lesser extent Cu, Hg and Zn) were highest in squid from Tasmania suggesting higher bioavailability of As (and potentially Cu, Hg and Zn) in the Tasman sea. Differences in trophic behaviors and diets of T. filippovae between locations, if they exist, could also explain some of the site-related differences in metal concentrations.

T. filippovae as a resource for man, a health viewpoint

As mentioned previously T. filippovae is likely to be an important part of the ecosystem as it is preyed upon by a number of vertebrates, including man. Moreover, stocks of more broadly targeted squid species undergo great fluctuations in abundance from year to year and will potentially be declining under an increasing pressure as global fisheries look to new populations to exploit [START_REF] Boyle | Cephalopods: Ecology and Fisheries[END_REF]. From this standpoint, T. filippovae could soon become a new targeted ressource and the object of much larger fisheries than it is today. Consequently, it is of interest to relate the metal concentrations given here to concentrations associated to health risks in humans.

Although they lack uniformity, guideline values are available for cephalopod consumers. The European Union has set regulations for Cd, Hg and Pb (European Commission, legislations 2001/22/CE) and the United Nations has set regulations for As in addition to the elements mentioned above. Silver, Cr and V are very seldom reported in mollusc studies and have not been appointed international legal concentration limits. International legal thresholds are also nonexistent for essential elements even though, in excess, these elements can be toxic.

Mercury is of particular concern for human health. High exposures to Hg, dependent on its form, can cause disruption of the nervous system, damage to brain functions, gastrointestinal tract and kidneys, DNA and chromosomal damage, allergic reactions and negative reproductive effects (such as sperm damage, birth defects and miscarriages) [START_REF] Clarkson | The toxicology of mercury -Current exposures and clinical manifestation[END_REF]. Inorganic mercury occurring naturally, or from pollution, is converted to methyl-mercury (MeHg) by microorganisms and is biomagnified up the food chain. Consequently, a large percentage of Hg is present as toxic MeHg in the edible portions of squids consumed by man. The most widely established guideline value for Hg concentrations in cephalopods is 0.5µg.g -1 ww. This value, as those that will be given hereafter for Cd, Pb and As, corresponds to the guideline value for whole gutted animals which, for squid, corresponds mostly to muscle tissue. We have thus compared only the muscular Hg concentrations to this recommended limit and found that they exceeded 0.5 µg.g -1 ww in 52% of cases in Ile Amsterdam and in 9% of cases in Tasmania. The theories according to which "exposure to MeHg exceeding guideline levels leads to adverse effects" or "maternal MeHg exposures are directly associated with adverse child development" should be considered with a critical eye as they are somewhat incomplete. First, as for any metal, unless the dose absorbed is extremely high, the exposure must be prolonged for adverse effects to appear. Second, the toxicity of a metal may be decreased by its interaction with other elements, such as Se in the case of Hg. Se is a nutritionally essential element that is present in all foods, but is particularly abundant in ocean fish and squid. A sufficient intake of Se offers a protective effect against Hg by their mutual irreversible binding [START_REF] Parizek | The protective effect of small amounts of selenite in sublimate intoxication[END_REF][START_REF] Iwata | Effect of selenium on methylmercury poisoning[END_REF][START_REF] Ohi | Efficacy of selenium in tuna and selenite in modifying methylmercury intoxication[END_REF][START_REF] Whanger | Selenium in the treatment of heavy metal poisoning and chemical carcinogenesis[END_REF]. For a detailed explanation of these biochemical processes refer to [START_REF] Ralston | Selenium health benefit values as seafood safety criteria[END_REF]. In order to better assess the true risk linked to Hg exposure, [START_REF] Kaneko | Selenium and mercury in pelagic fish in the central North Pacific near Hawaii[END_REF] have proposed a new measure of seafood safety that takes the protective role of Se into account. It is called the Selenium Health Benefit Value (Se-HBV) and is based on the relative quantity of Hg versus Se. Using this index, the authors conclude that most varieties of ocean fish (Hg:Se ratio around 1:5) are safe to eat. Squids also tend to be rich in Se. In this study, the large differences in the levels of the detection limits for Hg and Se prevent Hg:Se ratio comparisons in the muscle tissue. However, relatively large amounts of Se were measured in other organs: Hg:Se ratios in the digestive gland were 1:27 and 1:41, in Tasmanian and Amsterdam squid respectively, and around 1:3 in gills in both locations. The toxicity of Hg in the studied squid might thus be lesser than what appears from a simple comparison to consumption guidelines. The use of Se-HBV should be considered in further studies dealing with Hg toxicity in relation to squid consumption. It is however important not to forget that, although Se offers protection from the toxic effects of Hg, overexposure to Se can also cause damaging effects including serious neurologic, endocrine, and dermatologic effects (Agency for Toxic Substances and Disease Registry, 2003).

Cadmium is a stable, ubiquitous toxic metal, not abundant in its pure state in the environment. It mainly enters the humans organism via diet and inhalation (The Environmental Bureau of Investigation, 2009). Although the European Commission threshold concentration set for this metal in cephalopods is double that of Hg (1 µg.g -1 ww), Cd is also highly toxic (e.g. Jarup, 2003). It mainly causes kidney diseases but can also affect the liver, lungs, bone, immune system, blood and nervous system following chronic inhalation or oral exposure, and is possibly carcinogenic (United States Environmental Protection Agency, 2007b). In the muscle of T. filippovae all concentrations were under this guideline value. The 1 µg.g -1 ww: guideline value for Cd refers to gutted squid, hence without their digestive glands. However, cephalopods tend to accumulate Cd mainly in their digestive gland, and in greater concentrations than most other marine zoological groups [START_REF] Cognetti | Colonization of stressed coastal environments[END_REF]. Moreover, in Japan the digestive gland, called kimo, is essential for making a widely consumed dish named shiokara. It is thus noteworthy that in the present study 100% of digestive glands from both study sites showed higher concentrations than the authorized 1 µg.g -1 ww.

Lead can accumulate in bone and affects practically all systems within the body (e.g. central nervous system, kidneys and blood cells) (United States Environmental Protection Agency, 2007a). The guideline value adopted by the European Commission for Pb in cephalopods is 1 µg.g -1 ww. None of the individuals sampled had concentrations reaching this value. The Pb permissible value given above is more than 10 times higher than the maximum Pb concentration in muscle recorded during this study.

Arsenic is usually less of an issue in marine organisms than the metals previously discussed as, contrary to Hg, organic forms of As are less toxic than its inorganic forms. Marine organisms are able to convert inorganic As into organic As compounds [START_REF] Lunde | Occurrence and transformation of arsenic in the marine environment[END_REF]. In fact, numerous studies on animals have indicated that As is an essential nutrient although its physiological role is open to conjecture [START_REF] Uthus | Evidence for arsenic essentiality[END_REF]. Symptoms of acute intoxication in humans by inorganic As include severe gastrointestinal disorders, hepatic and renal failure, and cardiovascular disturbances, whereas chronic exposure causes skin pigmentation, hyperkeratosis, and cancers in the lung, bladder, liver, and kidney as well as skin [START_REF] Gorby | Arsenic in human medicine[END_REF]. The guideline value adopted by the World Health Organization for As in cephalopods is 2 µg.g -1 ww.. This value was not exceeded in the muscle of T.

filippovae from this study. Moreover, the figures from this study refer to measures of total As, part of which is likely to be present in a non-toxic form. In a recent study on market fishes and squids, [START_REF] Lin | Arsenic speciation in fish on the market[END_REF] found that 87% of As in muscle was arsenobetaine, a form of As which is non-toxic and non-carcinogenic to humans, and is rapidly excreted through urine after ingestion.

The dietary implications that stem from this study in terms of human consumption are the following. On one hand, As and Pb concentrations in T. filippovae caught around Ile Amsterdam or Tasmania are of no concern to human health. On the other hand, given the substantially high percentage (52%) of individuals from Ile Amsterdam accumulating muscular Hg past the health guideline concentration, a frequent and regular consumption of T. filippovae originating from this area is not recommended. Moreover, regardless of the squid's origin, digestive glands should be avoided as Cd and Hg concentrations were past the authorized limits in all, or nearly all, the digestive glands analyzed. It should also be mentioned that fetuses, infants, and children are more vulnerable to Hg, Cd, Pb and As exposure than adults since these metals are more easily absorbed into growing bodies, they pass biological barriers (e.g. into the brain) more easily and children's brains and nervous systems are more sensitive to their damaging effects. Parents should thus be particularly aware of the quantity of seafood products, including T. filippovae, eaten by their children, and pregnant women should of course closely monitor their diet.

Conclusion

This study of the bioaccumulation and tissue distribution of 14 trace elements in T. filippovae from the southern Indian and Pacific oceans has not only confirmed a number of trace element behaviors observed in other cephalopod species from distinct locations, but has also highlighted patterns specific to this species in these areas. The digestive gland and branchial hearts of T. filippovae contained the highest concentrations of most trace elements whereas the muscle tissue was characterized by the lowest concentrations, irrespective of where the squid were caught. This familiar pattern in trace elemental studies was somewhat contradicted here by the exceptions of two elements, As and Hg, that showed clear organotropisms towards muscle. Furthermore, the size of the squid was the most influential biological factor, followed by its geographic origin. Higher concentrations of Cd, Cr, Ni and Pb were noted in squid sampled in Ile Amsterdam compared to those caught in Tasmanian waters. Contrarily, concentrations of As were highest in squid from Tasmania. With respect to other cephalopod studies, metal concentrations of T. filippovae were overall within the spectrum of values found in cephalopods worldwide, often towards the lower end.

However in the comparison of metal concentrations to human health consumption guidelines the major conclusion is that T. filippovae squid from both, Tasmania and Ile Amsterdam, contain levels of Hg and Cd often exceeding the recommended guidelines, especially in their digestive glands.

Nevertheless, the non-negligible concentrations of Se also detected in these animals are to be kept in mind when considering Hg results because of the antagonistic effect of Se on Hg toxicity [START_REF] Parizek | The protective effect of small amounts of selenite in sublimate intoxication[END_REF][START_REF] Iwata | Effect of selenium on methylmercury poisoning[END_REF][START_REF] Ohi | Efficacy of selenium in tuna and selenite in modifying methylmercury intoxication[END_REF][START_REF] Whanger | Selenium in the treatment of heavy metal poisoning and chemical carcinogenesis[END_REF][START_REF] Ralston | Selenium health benefit values as seafood safety criteria[END_REF]. The toxicity of other non-essential elements such as Ag and Cd is also probably diminished by their binding to metallothioneins.

Acknowledgments

We are grateful to G. Duhamel, P. Pruvost, the crew of the research vessel La Curieuse and the crew of the Adriatic Pearl from Tasmania who participated in the sampling effort. The field work at Amsterdam was supported financially and logistically by the Institut Polaire Français Paul Emile Victor (IPEV, Programme N°558 109, H. Weimerskirch). The analytical investigations were supported financially by the LIENSs (University of La Rochelle) and by a Hermon Slade grant awarded to G.D. Jackson. We also thank the referees for their helpful comments on the manuscript.

Caption to figures Fig. 1. Levels of trace elements (µg.g -1 dw) in Todarodes filippovae tissues from Tasmania and Amsterdam (DG: Digestive Gland; BrH: Branchial Hearts). To assess whether the differences in concentrations were significant, one-way ANOVAs for paired samples were performed. The corresponding p-values are given above the boxplots.

Fig. 2 .

 2 Fig. 2. Continued from Figure 2. Levels of trace elements (µg.g -1 dw) in Todarodes filippovae

Fig. 3 .

 3 Fig. 3. Continued from Figure 3. Levels of trace elements (µg.g -1 dw) in Todarodes filippovae

Fig. 1

 1 Fig. 1

Fig. 2

 2 Fig. 2

Fig. 3

 3 Fig.3


Bassam Hajj

Sophie De Reguardati

Loïc Hugonin

Bruno Le Pioufle

Toshihisa Osaki

Hiroaki Suzuki

Shoji Takeuchi

Halina Mojzisova

Dominique Chauvat

Electro-Optical Imaging Microscopy of Dye-Doped Artificial Lipidic Membrane

Artificial lipidic bilayers are widely used as a model for the lipid matrix in biological cell membranes. We use the Pockels electro-optical effect to investigate the properties of an artificial lipidic membrane doped with nonlinear molecules into the outer layer. We report here what is believed to be the first electro-optical Pockels signal and image from such a membrane. The electro-optical dephasing distribution within the membrane is imaged and the signal shown to be linear as a function of the applied voltage. A theoretical analysis taking into account the orientation distribution of the inserted dye molecules allows to estimate the doped membrane nonlinearity. Future extensions of this work to living cell membranes are discussed.

Introduction:

As the main component of cellular membranes, lipid bilayers play an essential role as a barrier protecting the cell and its intracellular organelles. The cellular membranes also maintain the solute and ion concentration gradients which induce the equilibrium transmembrane potential. In the case of excitable cells, the plasma membrane ensures the propagation of the action potential. Various experimental techniques have been used in cellular electrophysiology for stimulation and measurement of membrane potentials by use of intracellular electrodes [START_REF] Hodgkin | Action potentials recorded from inside a nerve fibre[END_REF][START_REF] Llinas | Electrophysiological properties of in vitro Purkinje cell somata in mammalian cerebellar slices[END_REF], which has lead to the well known patch-clamp technique [START_REF] Sakmann | Patch Clamp Techniques for Studying Ionic Channels in Excitable-Membranes[END_REF]. Although these techniques are extremely sensitive and allow to record the electrical activity down to a single ion channel, they lead to cell death and provide the information about the potential in a single location within one neuron. Optical approaches using light scattering and intrinsic birefringence changes were investigated [START_REF] Cohen | Light scattering and birefringence changes during nerve activity[END_REF][START_REF] Cohen | Changes in axon birefringence during the action potential[END_REF]. Voltage-sensitive amphiphilic dyes were introduced to enhance the latter effect [START_REF] Gupta | Improvements in optical methods for measuring rapid changes in membrane potential[END_REF], and were found to show voltage-dependent change in absorption, dichroism [START_REF] Cohen | Optical methods for monitoring neuron activity[END_REF], and fluorescence [START_REF] Ross | Changes in absorption, fluorescence, dichroism, and Birefringence in stained giant axons: : optical measurement of membrane potential[END_REF][START_REF] Loew | Potentiometric dyes: Imaging electrical activity of cell membranes[END_REF]. Such electrometric dyes bind to the external cell membranes and contain an electron donor-acceptor group leading to electrochromism and a high hyperpolarizability. The membrane depolarization and the action potential propagation can then be recorded at different points of the cell as a voltage-induced fluorescence variation [START_REF] Zochowski | Imaging membrane potential with voltage-sensitive dyes[END_REF][START_REF] Zecevic | Multiple spike-initiation zones in single neurons revealed by voltagesensitive dyes[END_REF][START_REF] Gross | Dual-Wavelength Ratiometric Fluorescence Measurement of the Membrane Dipole Potential[END_REF], or by nonlinear microscopy techniques, in particular by second-harmonic generation (SHG) [START_REF] Dombeck | Optical recording of action potentials with second-harmonic generation microscopy[END_REF][START_REF] Bouevitch | Probing Membrane-Potential with Nonlinear Optics[END_REF][START_REF] Moreaux | Electrooptic response of second-harmonic generation membrane potential sensors[END_REF]. Voltage-induced SHG is a third-order nonlinear process described by an effective rank-four nonlinear susceptibility tensor associated to both quadratic and cubic timedependent molecular hyperpolarizability tensors. The former is underlying field induced orientational effects and the latter instantaneous four-wave mixing. Therefore SHG-based membrane potential measurements monitor the electrically induced changes of hyperpolarizability and/or the alignment of the dye molecules in the bilayer [START_REF] Bouevitch | Probing Membrane-Potential with Nonlinear Optics[END_REF][START_REF] Moreaux | Electrooptic response of second-harmonic generation membrane potential sensors[END_REF]. A major interest of nonlinear microscopy lies in its intrinsic three-dimensional sectioning capability related to the two-photon excitation step that occurs mainly in the focal volume together with the low photobleaching of out-of-focus molecules. It should be however noted that the bleaching may be increased in the focal spot due to the use of pulsed femtosecond lasers. Remarkably, the non-centrosymmetric organization of the dye molecules, inserted exclusively into the external membrane layer and responsible for SHG, is also at the origin of the related electro-optical Pockels effect [START_REF] Toury | Electro-optical microscopy: mapping nonlinear polymer films with micrometric resolution[END_REF]. The Pockels effect is a variation of the index of refraction induced by a quasi-static applied electric field. When probed by a laser beam, induces an optical phase shift which can be detected with an interferometric setup. Since the Pockels effect is a non-resonant process, it can be measured using a low-power continuous wave laser, which preserves the integrity of the sample, and more generally avoids the use of costly and cumbersome high power short pulse lasers. In this work we present an original electro-optical microscopy study of the variation of membrane potential in model lipid bilayers stained by the potentiometric Di-8-ANEPPS dye. We measure the absolute change in index of refraction due to electro-optical effect along a chosen polarization axis. Since lipid bilayers are considered to be an adequate model for the investigation of the electric properties of biologic membranes [START_REF] Pons | Mechanisms of membrane potential sensing with second-harmonic generation microscopy[END_REF], electro-optical measurements were subsequently performed on a phosphatidylcholine bilayer formed on a parylene biochip. Our results demonstrate the imaging capability of an advanced electrooptical microscope in a microfluidic environment suitable for biology-related studies.

Principle and theory:

The electro-optical (EO) effect of a non-centrosymmetric material, or Pockels effect, is the change of the index of refraction of this medium in response to a quasi-static electrical field applied using adequately shaped electrodes [START_REF] Boyd | Nonlinear Optics[END_REF]. The index of refraction change can be read out as an additional small phase shift on a probing light wave. While the Pockels effect stems from a second-order (rank three) nonlinearity, it can also be viewed as a linear optical response in the electric field of the probe light field op , which also depends linearly on the applied external quasi-static electrical field . The index of refraction change along the i axis is given by ,

where E j is the component of the applied electric field in the j direction (See Appendix A). The proportionality factors r ij are the coefficients of the electro-optical tensor [START_REF] Boyd | Nonlinear Optics[END_REF]. We choose {x,y,z} as the principal axes of the nonlinear material, i.e., perpendicular to the plane of the doped bilayer (see Fig. 1). z is along the applied field direction and x,y are arbitrary directions in the sample plane. Depending on the orientation of the molecules inside the medium and the associated geometrical symmetry, some r ij coefficients cancel and some are related one to the other. In the case of an artificial bilayer doped with an ensemble of dye molecules, the cylindrical symmetry around the z axis leads to the following tensor (see Appendix A) .

(

) 2
In this notation index '3' is related to the z direction of propagation of the light, perpendicular to the bi-membrane (see Fig. 1). The induced EO phase shift is ,

that can be developed as .

A signature of the effect can be obtained by rotating the polarization of the linearly polarized incident light. Inspection of the electro-optical tensor matrix shows that the phase shift must not change when the polarization of the incident beam is rotated within the (x,y) plane of the sample, as it involves the same r 13 EO coefficient, in agreement with the cylindrical symmetry about the z axis. We therefore note that a polarimetric detection setup with crossed polarizer and analyser, which is sensitive to a differential phase shift between two orthogonal directions, would not detect the above isotropic phase shift. An optical scheme capable of measuring the absolute EO phase shift along one given polarization direction is thus required. Such scheme could measure the electrical field if the EO coefficients are known, or conversely the EO coefficients can be extracted when the electric field is known. If both are undefined, it can access to the product. Assuming a potential of 10mV throughout a membrane of e=7nm thickness and a realistic r coefficient of the order of 1pm/V for a doped membrane, one should be able to measure a corresponding phase shift variation of the order of 10 7 radian. In order to measure this additional EO dephasing contribution, we use an interferometric scheme described below.

Sample and optical methods:

3.a Electro-optic microscope:

The principle of measuring an electro-optical phase shift is shown in Fig. 2. A continuous wave He-Ne laser is used as the source of light, its beam being subsequently separated in two.

One part is focused on the doped lipidic membrane through a microscope objective (Nikon , CFI plan-fluor 40 , N.A. = 0.6), then transmitted and collected via an identical microscope objective in forward direction. It is then recombined with the other beam that plays the role of the reference arm in a Mach-Zehnder interferometric scheme [START_REF] Born | Principles of Optics -Electromagnetic Theory of Propagation, Interference and Diffraction of Light[END_REF]. The interference optical signal is detected by two photodiodes (Hamamatsu). The electric field applied on the sample is typically modulated at a frequency kHz, inducing a modulated phase shift and interference fringes changes at the same frequency. To reach a high sensitivity for detecting a small phase shift variation, a balanced homodyne detection based on two photodiodes is being used [START_REF] Le Xuan | Balanced homodyne detection of secondharmonic generation from isolated subwavelength emitters[END_REF]. A lock-in amplifier (EG&G Princeton Applied Research, Model 5302) is used to measure this modulated phase shift with high signal-to-noise ratio. The sensitivity of the interferometric detection to the EO phase shift is maximized if the static optical path difference between the two beams corresponds to a phase shift rad. A phase control loop is therefore implemented in the system, with a mirror mounted on a piezoelectrical transducer stage (PI ceramic). It is added to the optical path of the reference beam and its translation allows to change the quasi-static optical path length (not shown in Fig. 2). Let us note that in principle an electrically-induced absorption modification would also lead to a signal modulated at frequency . However, with the above phase working point , the lock-in amplifier is sensitive only to the EO phase-shift, while would render the detection system sensitive to a modulated absorption (See Appendix C). Two half-wave plates (Fichou, France) allow to control the polarization of the light focused onto the sample.

3.b Sample composition and fabrication: Bio chip:

As a sample holder, we use a home-made bio-chip made of parylene. It is encapsulated in a resist that has been photopatterned in three dimensions thanks to a stereolithography process [START_REF] Suzuki | Ninety-six-well planar lipid bilayer chip for ion channel recording fabricated by hybrid stereolithography[END_REF]. The parylene thin film of 20µm thickness was micromachined using O 2 plasma in reactive-ion-etching through an aluminum mask, which leads to an array of trough holes devoted to the bilayer reconstruction. In each microchamber, 9 holes of diameter 40 µm allowed the simultaneous formation of 9 bilayers [START_REF] Le Pioufle | Lipid bilayer microarray for parallel recording of transmembrane ion currents[END_REF][START_REF] Suzuki | Highly reproducible method of planar lipid bilayer reconstitution in polymethyl methacrylate microfluidic chip[END_REF][START_REF] Ide | A novel method for artificial lipid-bilayer formation[END_REF]. Electrodes: Two Ag/AgCl electrodes, connected to the solutions from each side of the bilayer, were used to apply a voltage difference. The coating of silver electrodes is deposited by dipping the electrodes for a few minutes in a nitric and hydrochloric acid (ROTH) (1/3 vol.vol) concentrated solution. By dipping the electrodes in this bath for a few minutes, we obtained a pure, thin and uniform chloride silver coating.

3.c Mechanism of bilayer formation:

Prior to this experiment, the bio-chip is first put into an ultra-sound bath in ethanol solution for 5 minutes, and then in another ultra-sound bath in Millipore water solution for 2 minutes. It was rinsed with Millipore water, dried with compressed nitrogen, then in an oven (40°C, 10mn). 15µl of a buffer solution of KCl 0.1M (Fisher Scientific) and MOPS (3-(Nmorpholino)propanesulfonic acid) 10mM pH7 (Research Organics) was inserted in the upper chamber (see Fig. 3.a). The lipid used was L-α-phosphatidylcholine (Egg, Chicken) (Avanti Polar Lipids), 20 mg/ml dissolved in N-decane (Merck). 8µl of the lipid solution was flowed in the lower channels (see Fig. 3.b), followed by the flowing of buffer, which permits the formation of the bilayers (see Fig. 3.c), as described in Refs. [START_REF] Suzuki | Ninety-six-well planar lipid bilayer chip for ion channel recording fabricated by hybrid stereolithography[END_REF][START_REF] Le Pioufle | Lipid bilayer microarray for parallel recording of transmembrane ion currents[END_REF].

Staining of the bilayer:

To generate an electro-optical phase shift the medium has to display a non-centrosymmetric statistical arrangement. The lipid molecule by itself satisfied this condition, but in the case of a bilayer the head-tail molecular assembly leads to a centrosymmetric system, and subsequent cancellation of the EO effect. In order to confer electro-optical activity to the membrane we asymmetrically stained the membrane with a non-centrosymmetric probe molecule Di-8-ANEPPS (Invitrogen). This molecule has a long carbon chain, thus adequately lengthening the flip time across the membrane [START_REF] Loew | Potentiometric dyes: Imaging electrical activity of cell membranes[END_REF][START_REF] Rohr | Multiple-site optical-recording of transmembrane voltage (Msortv) in patterned growth heart cell-cultures -assessing electrical behavior, with microsecond resolution, on a cellular and subcellular scale[END_REF]. Those properties make Di-8-ANEPPS a dye that has been widely used for fluorescence or SHG measurement of membranes [START_REF] Bullen | High-speed, random-access fluorescence microscopy: II. Fast quantitative measurements with voltage-sensitive dyes[END_REF][START_REF] Ries | Black lipid membranes: visualizing the structure, dynamics, and substrate dependence of membranes[END_REF], and as an electrical potential probe [START_REF] Loew | Potentiometric dyes: Imaging electrical activity of cell membranes[END_REF][START_REF] Gross | Dual-Wavelength Ratiometric Fluorescence Measurement of the Membrane Dipole Potential[END_REF][START_REF] Rohr | Multiple-site optical-recording of transmembrane voltage (Msortv) in patterned growth heart cell-cultures -assessing electrical behavior, with microsecond resolution, on a cellular and subcellular scale[END_REF][START_REF] Antic | Optical signals from neurons with internally applied voltage-sensitive dyes[END_REF][START_REF] Clarke | Effect of lipid structure on the dipole potential of phosphatidylcholine bilayers[END_REF][START_REF] Clarke | Optical detection of membrane dipole potential: Avoidance of fluidity and dye-induced effects[END_REF][START_REF] Shynkar | Two-color fluorescent probes for imaging the dipole potential of cell plasma membranes[END_REF][START_REF] Zhang | Membrane electric properties by combined patch clamp and fluorescence ratio imaging in single neurons[END_REF]. Its orientation in the membrane was investigated [START_REF] Ries | Black lipid membranes: visualizing the structure, dynamics, and substrate dependence of membranes[END_REF][START_REF] Lambacher | Orientation of hemicyanine dye in lipid membrane measured by fluorescence interferometry on a silicon chip[END_REF], and different models were proposed. The stock solution was prepared in DMSO (Sigma Aldrich) at 1mg/ml concentration. 3µl of the dye was introduced to the upper chamber already filled with the buffer just after the injection of the lipid in the lower channel. Then a glass plate was placed on the bio-chip to cover the upper chamber. It was used to prevent the formation of a curved liquid surface that could act as a micro-lens and thus affect the probe laser beam that passes through it. The glass plate was perforated to insert an electrode inside the buffer solution. Then, as explained above the buffer was introduced in the lower channel and the bilayer formed. A 15-minute waiting time between the introduction of the dye and the start of the measurement allowed the dye to intercalate into the upper layer of the artificial bilayer. In order to check that the lipidic membrane was organized as a bilayer, a capacitance measurement between both sides of the membrane was made before and after the electrooptical measurement described below. The same capacitance 0.32 µF/cm 2 measured at the beginning of the experiment was found also at the end. This is in good agreement with the values of surface capacitance of bilayers (0.4 µF/cm 2 ) reported in the literature (34).

Results:

Once a stable bilayer was formed in the bio-chip, we were able to stain the membrane and then perform the electro-optical measurement. The dye was inserted on one side of the lipidic membrane, and a modulated electric field was applied between both sides of this membrane. A sinusoidally-varying potential difference up to 10 Volts maximum amplitude was applied to the membrane without breaking it, and its electro-optical response was registered. The modulated electric field induced a modulated phase shift in the laser beam that probes the EO active sample. Though the signal was small, our interferometric setup proved sensitive enough for measuring this electro-optical phase shift through the membrane. First the electro-optical signal was detected with a good signal-to-noise ratio (SNR). For a voltage difference of 5V peak-to-peak amplitude at 100 kHz, the SNR was 10 ± 1 for 20 ms integration time. Second we observed that the temporal response of the system was fluctuating (Fig. 4). This may be due to impurities inside the buffer passing through the probe beam during the measurement. The frequency of the modulated electric field applied to the membrane is limited by the cutoff frequency of the whole system. The bilayer and the surrounding buffer can be modeled as a capacitor and a resistance [START_REF] Hall | Access resistance of a small circular pore[END_REF]. We estimate a cut-off frequency about 3 MHz, well above the frequency used for the measurement. As for the conductivity of the buffer necessary to transfer the voltage difference to the membrane at a given frequency, we calculated a cut-off frequency of 260 MHz. In order to check the origin of the detected signal, we changed the amplitude of the applied potential difference. Temporal signal of the lock-in amplifier was recorded for each modulated voltage amplitude, and the dependence of the EO signal on the applied voltage was plotted. Fig. 5 shows a linear dependence, as expected for the Pockels effect (from Eq.1).

From the slope of this curve we could extract the electro-optical coefficient of the membrane. The slope is (43.8 ± 1.7) 10 6 rad/V, which is theoretically equal to (see Eq. 18 in Appendix). The laser wavelength is 632.8 nm. It is difficult to give accurate values to the thickness and index of refraction of the membrane for the electro-optical response. Here we make the assumption that a macroscopic description of this ultra-thin optical device is valid, which is commonly done for thin films [START_REF] Zochowski | Imaging membrane potential with voltage-sensitive dyes[END_REF]. Under this assumption, e 7nm and n 1.5 seem to be safe values, which have been used in the literature [START_REF] Lis | Interactions between neutral phospholipid-bilayer membranes[END_REF]. Then one can estimate the membrane EO coefficient =2.6 pm/V. This value seems reasonable since it can lead to non-negligible SHG signals as those reported in ref [START_REF] Ries | Black lipid membranes: visualizing the structure, dynamics, and substrate dependence of membranes[END_REF]. Fig. 6 shows a typical raster scan of a membrane under a 5V peak-to-peak sinusoidal voltage at 20 kHz frequency. The contour of the hole holding the membrane can be distinguished. The signal is higher at the center of the membrane, maybe due to a higher density of Di-8-ANEPPS molecules at the center of the hole, itself due to a possible Gibbs plateau on the circumference of the hole. The inset on Fig. 6 shows another scan during which the membrane was broken. In order to test the symmetry of the dye orientation distribution, the linear polarization of the incoming light was switched perpendicular to the previous one, i.e, from x to y axis in Fig. 1. The results are given in Tab. 1. The signal did not change within 2%, as expected from the cylindrical symmetry about the z axis (see Fig. 1), hence the same electro-optical coefficient is found in both configurations. Again we note that this isotropic phase shift distribution of the EO variation of the index of refraction could not be detected by a standard polarimetric setup sensitive to birefringence. We compared such a response to a home-made reference sample built with a LiNbO 3 single crystal plate (see Tab.1). This reference is highly anisotropic in its x and y responses, which confirmed the polarization sensitivity of our interferometric detection method. Therefore any change of dye alignment in the membrane can be readily detected by our set-up.

Discussion and Conclusion:

In conclusion we demonstrated that the electro-optical effect can be used to quantitatively image a dye-doped artificial membrane. We checked that the EO response is proportional to the applied voltage difference as expected for the Pockels effect. Using different optical polarization, we also checked the symmetry of the doped membrane around the direction normal to the bilayer, and proved that this microscopy is able to probe the statistical orientation order of the dye inside the membrane. We point out that this interferometer is sensitive to absolute electro-optical variation of the index of refraction along one selected polarization direction. Estimating the applied electrical field we obtained a reasonable value for the dye-doped membrane nonlinear susceptibility =2.6pm/V. If the nonlinear susceptibility is known independently, for instance from SHG measurement, our method provides a non-resonant optical measurement of the transmembrane potential. This second approach therefore allows to investigate electrical fields in doped lipid membrane. We note that the above results rely on average values over the membrane. A future analysis taking into account the distribution of the field and exact molecular insertion could be performed by using different dye molecules. At the scale of a single layer of molecules, we may also observe heterogenous physical effects, in terms of thin-film organization and for multicomponent membranes which will require a refined model. We note that a modulated sinusoidal potential difference of 1 Volt amplitude at a relatively high frequency varied from 15 to 100 kHz, was not invasive to the membrane. For an unstained membrane the voltage reached 4 Vpp at frequency = 20 kHz before breaking the membrane, in the case of an electrical capacitance of 33 pF for 9 simultaneous bilayers. With stained lipidic membranes, some of them could withstand a voltage application up to 20Vpp at 20 kHz, without damage. This observation is quite surprising since it is well known that when a DC potential difference higher than 1V is applied to a bilayer, it is depolarized by the corresponding static electric field and the membrane is broken [START_REF] Crowley | Electrical breakdown of biomolecular lipid-membranes as an electromechanical instability[END_REF][START_REF] Kramar | Determination of the lipid bilayer breakdown voltage by means of linear rising signal[END_REF][START_REF] Lebar | Inter-pulse interval between rectangular voltage pulses affects electroporation threshold of artificial lipid bilayers[END_REF]. The enhanced robustness of the membrane to higher voltage is probably linked to the sinusoidal character of the potential difference. This unexpected result has to be investigated in future work. Many developments of the technique can now be envisioned. For instance, in order to fully characterize the orientation order of the dye in the artificial bilayer (see Fig. 7), the average tilt angle of the molecule being given by the coefficient ratio (see Appendix Eq.15), a light polarization in the z axis direction could be applied on the membrane by focusing an inhomogeneously polarized beam [START_REF] Dorn | Sharper focus for a radially polarized light beam[END_REF]. Furthermore new bio-chips where the flow of buffer solution in the upper chamber may be modified can also be designed. It would allow us to change the dye concentration during a single experimental measurement and also to insert membrane proteins that have the potential to control membrane channel opening and the related ionic concentration that are responsible for membrane potential variations. Finally, it can also be envisioned to probe the depolarization of plasma membrane after stimulation and in long term the propagation of an action potential in an excitable cell. Some bottlenecks have however to be considered. First, such internally generated electrical field is a self-triggered train of pulses rather than a sine wave at a definite frequency. Therefore a new asynchronous detection technique has to be developed. The pulse repetition rate being also low, an heterodyne detection would be a good candidate to shift low frequency to a higher noise-less one [START_REF] Agrawal | Fiber-Optic Communication Systems[END_REF]. Second, in the case of a living cell the surface is not flat thus leading to a tilt angle of the normal direction to the membrane with respect to the microscope Z-axis at the probe beam position (see Fig. 8). In this case new electro-optical coefficients will appear (see Appendix C Eq. 22). A simultaneous polarization measurement in two perpendicular polarizations could measure this local tilt . Third, for thick biological sample a transmission mode clearly constitute a limitation. A reflection one could be used since the index of refraction variation under an applied electric field induces a variation of the reflection coefficient of the membrane. Using Abeles matrix in stratified medium [START_REF] Born | Principles of Optics -Electromagnetic Theory of Propagation, Interference and Diffraction of Light[END_REF], the expected intensity reflection coefficient is approximately 10 -5 in intensity, and the electro-optical contribution is estimated to be .A modified setup with enhanced sensitivity is envisioned to detect this small effect. While the ultimate goal of applying our technique to the electrical activity of a living cell, under investigation in our lab, is challenging, the results presented here are promising to further explore an all-optical patch-clamp technique.

Acknowledgments:

We thank Jean-Pierre Lefèvre and Jean-Pierre Madrange for technique assistance. We acknowledge contract CNano MELOA and F-I Bio-imaging.

32 Tab. 1 :Tab. 2 :Fig. 1 :Fig. 2 :Fig. 3 :Fig. 4 :Fig. 5 :Fig. 6 :Fig. 7 :Fig. 8 :

 321212345678 Fig. 1 : Principle of probing the electro-optical effect of a one-side dye-doped membrane. Inset: orientation distribution of the dye molecules in the upper layer.

APPENDIX A. Relation between the nonlinear susceptibility and molecular orientation:

The electro-optical (EO) effect is the change of the refractive index of a medium in response to a quasi-static electrical field. This effect is related to the second-order nonlinearity and the related EO susceptibility is given by : ,

where is the nonlinear second-order susceptibility, is the optical frequency, is the applied electrical field frequency which can be assumed to be quasi-static compared to that of the optical frequency. N is the density of non-linear chromophores and is their molecular hyperpolarizability, is the solid angle related to the orientation of the molecules, as defined for each molecule by its set of Euler angles. and are the local field factors at optical frequency and quasi-static frequency. Referring to x,y,z laboratory frame, i,j,k are the macroscopic coordinates. The tensor of the molecule is known and simple in the axes X,Y,Z associated to the geometry of the molecule. It can be assumed to be one-dimensional, i.e.

, in agreement with the quasi 1-D structure of the dye and related intra-molecular charge transfer that underlies tensor. The associated coordinates are I,J,K. Taking into account the transformation from X,Y,Z to x,y,z for laboratory axes, Eq. 5 becomes:

.

In the case of a bilayer having a thickness e, doped by a nonlinear dye with a surface density N s we have . We assume that the molecules are oriented inside the membrane in a cone with a symmetry around the normal z to the membrane surface as shown in Fig. 1 and Fig. 7 , being the angle of this cone with respect to the normal. In the case of a dye oriented inside a membrane in first approximation, one can express the probability of orientation as:

, therefore:

This cylindrical symmetry leaves only two independent non-zero coefficients of the tensor, namely: and

,

and .

Electro-optic coefficients: The electro-optical coefficients are related to the second-order susceptibility [START_REF] Sigelle | Determination of the electrooptic coefficients of 3methyl 4-nitropyridine 1-oxide by an interferometric phase-modulation technique[END_REF], by the relation

We can write those coefficients in the contracted indices mode where ijk will become lk and l stands for a combination of ij [START_REF] Yariv | Optical Waves in Crystals -propagation and control of laser radiation[END_REF]. It follows the Tab. 2.

From the above geometry and dye characteristics, the electro-optical coefficient tensor matrix of our doped artificial bilayer takes the form: ,

where

and

These expressions show the dependence of the electro-optical coefficient on the orientation angle of the dye. The ration between the two coefficients is directly linked to this angle:

EO induced phase shift:

The additional EO phase shift across a membrane of thickness e, when the beam is polarized along the direction, can be expected as: ,

,

where E j is the applied quasi-static electric field.

In the configuration where the applied electric field is in the direction normal to the membrane i.e. along z, the only nonzero component of the electrical field is E 3 , then : ,

,

.

B. Case of biological membrane:

In the case of a cell membrane, the surface is not horizontal and the morphology is more complex. The simplest model is a bilayer with a tilt angle with respect to the horizontal line (see Fig. 8). In this configuration the new electro-optical coefficients, read in the principal axis system (1',2',3'), can be expressed as a function of the coefficient, in the membrane axis system (1,2,3). They can be written:

(21) In the new axis system the EO coefficient tensor will be then given by: [START_REF] Le Pioufle | Lipid bilayer microarray for parallel recording of transmembrane ion currents[END_REF] The new phase shift under two perpendicular polarizations can be written: , [START_REF] Suzuki | Highly reproducible method of planar lipid bilayer reconstitution in polymethyl methacrylate microfluidic chip[END_REF] .

(24) A dependency on the tilt angle is clearly expected.

C. Homodyne and synchronous detection:

If a reference beam of amplitude is interfering with a signal beam of amplitude , the difference in the intensities detected by the two photodiodes used in balanced homodyne detection configuration is:

(25) where is the static optical phase-shift between the two beams paths, and the electrooptical phase-shift in the signal path. While we are interested in measuring one can wonder whether a signal amplitude variation due to absorption or emission could contribute to the signal.

Two regimes are of interest depending on the static phase shift value. First, if , eq. 25 became provided that the electro-optical phase shift is small. The electric field being modulated at a frequency , the EO phase shift will be modulated at the same frequency ( ) as well as the emission or absorption .Then . (26) The synchronous detection multiplies by a reference signal at the electric field frequency . After such multiplication the DC component carries the information of the EO phase shift corresponding to the first term in equation [START_REF] Bullen | High-speed, random-access fluorescence microscopy: II. Fast quantitative measurements with voltage-sensitive dyes[END_REF] . The two last components are shifted spectrally to higher frequency, which includes the intensity variation due to absorption or emission variation. Therefore will not show-up in the DC expression.

Second, if

; then the intensity difference becomes if is very small. In this case any variation in the amplitude of the signal due to absorption or emission induced by electric field and modulated with the same frequency will be measured with the lock-in amplifier.

Therefore an interesting feature of our method is to be able to access either EO coefficient or electric field-induced absorption and emission variation, depending on the phase shift that we impose between the two interfering beams we can.

, , .