C.-H Hage

F Billard

B Kibler

C Finot

G Millot

Direct temporal reconstruction of a picosecond pulse by cross-correlation in a semiconductor device

Cross-correlation measurements using the two-photon absorption process in a semiconductor is experimentally demonstrated for two pulses of different wavelengths (shifted by ~200 nm) and durations (20 times ratio). These measurements were found to be highly repeatable and fully suitable for the determination of the temporal intensity profile of picosecond (ps) pulses.

Introduction: Ultrashort optical pulses are nowadays a basic requirement for numerous applications such as optical communications, nonlinear spectroscopy or terahertz field [START_REF] Kaizer | Ultrashort laser pulses, generation and applications[END_REF]. As measurement and control of these pulses cannot be achieved by electronic means, alloptical methods have been developed. For example, temporal intensity profiles can be reconstructed from cross correlation records based on an interferometric device which measures the correlation between an unknown pulse and a reference ultrashort pulse through their product in a nonlinear crystal. Although these methods are accurate, a delicate trade-off has to be found between efficiency and spectral bandwidth [START_REF] Ranka | Autocorrelation measurement of 6-fs pulses based on the two-photon-induced photocurrent in a GaAsP photodiode[END_REF]. This drawback can be partly circumvented by using the two-photon absorption (TPA) process in a semiconductor which permits wideband and ultrashort pulses measurements [START_REF] Ranka | Autocorrelation measurement of 6-fs pulses based on the two-photon-induced photocurrent in a GaAsP photodiode[END_REF][START_REF] Laughton | The two-photon absorption semiconductor waveguide autocorrelator[END_REF][START_REF] Krug | High-Sensitivity Two-Photon Absorption Microcavity Autocorrelator[END_REF], the process being only limited by the peak power of the pulses [START_REF] Krug | High-Sensitivity Two-Photon Absorption Microcavity Autocorrelator[END_REF].

Optical cross-correlation between pulses widely different in terms of central wavelength and pulse duration (∆λ > 100 nm and ∆T ratio > 10) has been demonstrated in nonlinear crystals [START_REF] Deng | Experimental research on measuring the fine structure of long pulse in time domain by synchronized ultrashort pulse[END_REF] but to the best of our knowledge, it has never been reported in a semiconductor device. Here we present what we believe to be the first cross-correlation between two completely different pulses in a semiconductor device. Experimental setup and results: The experimental setup is depicted in Fig. 1. A femtosecond (fs) laser (Amplitude Systèmes tPulse model, central wavelength of 1033 nm, pulse duration of 180 fs, repetition rate of 49.6 MHz, average power of 1 W) is divided in two by a beamsplitter (BS). In the first part of the setup, wavelength tunable pulses are created using the Raman soliton self-frequency shift (SSFS) process [START_REF] Mitschke | Discovery of the soliton selffrequency shift[END_REF] occurring in a highly non-linear photonic crystal fibre with anomalous dispersion. These pulses are Fourier transform limited and have a nearly-constant full-width at half-maximum (FWHM) duration τs = 120 fs (details of the experimental implementation and results of the SSFS block are provided in [START_REF] Hage | Optimization and characterization of a femtosecond tunable light source based on the soliton self-frequency shift in photonic crystal fiber[END_REF]). A long-pass filter (LPF) is used to remove the pump residue. A second block of the setup is based on the spectral compression (SC) process [START_REF] Planas | Spectral narrowing in the propagation of chirped pulses in singlemode fibers[END_REF] that enables the generation of ps pulses with a spectral width of ~1 nm (such a spectral narrowing is experienced upon the nonlinear propagation in optical fibre of chirped pulses, details of the block are not provided here). These two pulses are spatially recombined by a dichroic beam-combiner (BC) and their temporal overlap is controlled by means of an optical delay line (DL).

The pulses are collinearly polarized in order to avoid the influence of the anisotropy of the photodiode semiconductor nonlinear absorption [START_REF] Santran | Characterization of Optical Nonlinearity in Semiconductor Photodiodes Using Cross-Polarized Autocorrelation[END_REF] and are tightly focused on a GaAsP photodiode (PD, Hammamatsu G1116 model). The resulting photocurrent is monitored by an oscilloscope with high input impedance (1 MΩ). In order to get a reasonable optical signal to noise ratio, we used pulse energies of about 30 pJ although lower energy levels (10 pJ) are sufficient.

We first study cross-correlation measurements involving ultrashort solitons shifted (by SSFS) to 1150 nm. The resulting signal is shown in Fig. 2a. As the fs pulse is more than one order of magnitude shorter than the ps one, it can be in first approximation considered as a Dirac function for the convolution operation. In other words, the crosscorrelation record should reproduce directly the temporal intensity profile of the ps pulse [START_REF] Kaizer | Ultrashort laser pulses, generation and applications[END_REF]. We have checked this assumption by computing autocorrelation signal when considering the intensity profile of the ps pulse being equal to the cross-correlation signal (Fig 2b, circles) and we have experimentally compared the result with an independently measured autocorrelation signal (solid line, measurements made with a Femtochrome FR-103HS device based on second harmonic generation in a nonlinear crystal). The two autocorrelation signals have been found in excellent agreement, which validates that given the very different temporal durations under investigation, there is no need of carrying out complex deconvolution processes to take into account the temporal extend of the shortest pulse. We can therefore estimate directly the FWHM of the pulse, i.e. Auto-correlation computed from the temporal intensity profile inferred from the cross-correlation record (circles) and compared with an independently measured autocorrelation signal of the ps pulse.

In Fig. 3a we further check that the fs pulse can be considered as a Dirac function for the cross-correlation operation. We numerically simulated cross-correlation signals between the ps pulse and pulses of different durations (from 1 ps down to 10 fs) and in order to quantify our error, we compute two relevant parameters. The first one is the error made in estimating the FWHM duration directly from the crosscorrelation signal. We can see that a fs pulse duration of less than half the ps one leads to an error below 5 %, which means that the duration parameter is not critically affected by the ratio between the pulses durations (as long as the intensity profiles under test are not too complex and do not present ultrashort substructures). The second parameter we use is a misfit parameter [START_REF] Finot | Parabolic pulse evolution in normally dispersive fiber amplifiers preceeding the similariton formation regime[END_REF] aimed at quantifying the error between the temporal intensity profile I(t) of the long pulse and the cross-correlation signal X(t). It can therefore be defined as :

M 2 = ∫[I(t)-X(t)] 2 dt/∫I(t) 2 dt (1)
We can make out from Fig. 3a that this parameter is much more sensitive than the FWHM duration: pulse durations less than τp/10 are required in order to reach a 5 % misfit. As our fs pulse is about τp/20 it is far short enough to fit our needs. Finally to assess the "broadband" aspect of our setup we made crosscorrelation measurements for different fs pulse wavelengths (the femtosecond pulse wavelength is easily tuned by varying the optical peak power at the input of the SSFS block [START_REF] Mitschke | Discovery of the soliton selffrequency shift[END_REF][START_REF] Hage | Optimization and characterization of a femtosecond tunable light source based on the soliton self-frequency shift in photonic crystal fiber[END_REF]). The results are shown in Fig. 3b and exhibit as expected a nearly constant pulse duration. Note that our method is here limited by the maximum redshift of the SSFS block. By using the same photodiode, the TPA process is expected to work with ultrashort shifted solitons up to 1700 nm.

Conclusion:

We have experimentally demonstrated direct and broadband temporal reconstruction of picosecond pulses based on crosscorrelation measurements in a semiconductor device. By considering pulses with quite distinct temporal durations we overcome the need of deconvolution algorithms and the required details of the shortest pulse. In our configuration where the ps pulse under investigation does not present rapid fluctuations, the cross-correlation can indeed be considered as reproducing directly the temporal intensity profile of the longer pulse. The method has been found robust compared to the wavelength spacing between the two pulses and the measurements have been highly repeatable. It can be considered as a simple, clean, reliable and highly integrable multipurpose tool to synchronize beams, measure jitters or doing accurate picosecond pulse measurements.

Fig. 1

 1 Fig. 1 Experimental setup. BS = Beam-Splitter, SSFS = Soliton Self-Frequency Shift, SC = Spectral Compression, LPF = Long Pass Filter, DL = optical Delay Line, BC = Beam Combiner, PD = PhotoDiode

Fig. 2

 2 2.2 ps, without assuming any shape factor as usually required for measurements based on autocorrelation signals. The two independently measured signals have also confirmed that no temporal substructure was present in the picosecond pulse. a b Cross-correlation results. a Cross-correlation signal (result is averaged over 10 independent measurements) b

Fig3

  Direct and broadband temporal reconstruction. a Numerical analysis: influence of the ratio of pulse durations involved in the cross-correlation. Error on the FWHM temporal duration of the ps pulse (circles, right axis) and misfit parameter (crosses, left axis). The vertical dashed line indicates the ratio used in our experiments. b Experiments: temporal pulse duration (FWHM) obtained from crosscorrelation measurements for different fs pulses wavelengths.

Acknowledgments: We acknowledge the support of the Agence Nationale de la Recherche (ANR SOFICARS) through the grant ANR-07-RIB-013-03. C.-H. Hage acknowledges the support of the Conseil Régional de Bourgogne through his grant "Jeune Chercheur Entrepreneur" scheme. B. Kibler also thanks the support of the Conseil Régional de Bourgogne through the Photcom PARI grant. C.-H. Hage, F. Billard, B. Kibler, C. Finot and G. Millot (Laboratoire Interdisciplinaire Carnot de Bourgogne, UMR 6303 CNRS-Université de Bourgogne, Dijon 21078, France) E-mail: charles-henri.hage@u-bourgogne.fr


Patrice Bertrand

Jean Diatta

Correspondences between Pre-pyramids, Pyramids and Robinsonian Dissimilarities

Keywords: Cluster structure, Compatible order, Robinsonian dissimilarity

We consider cluster structures in a general setting where they do not necessarily contain all singletons of the ground set. Then we provide a direct proof of the bijection between semi-proper robinsonian dissimilarities and indexed pre-pyramids. This result generalizes its analogue proven by Batbedat in the particular case of definite cluster structures. Moreover, the proposed proof shows that the clusters of the indexed pre-pyramid corresponding to a semi-proper robinsonian dissimilarity are particular 2-balls of the considered dissimilarity.

Structural bijections between cluster structures and dissimilarity types play an important role in cluster analysis. They not only validate the dissimilarity-based clustering approach, but make it possible to bring a cluster system specification problem back to a dissimilarity matrix determination (13; 9; 8; 10). The best known of these bijections is certainly the one between indexed hierarchies and ultrametrics (20; 5). Several extensions of this bijection have been obtained, e.g., between indexed quasi-hierarchies and quasi-ultrametrics (1; 15), between indexed pyramids and strongly robinsonian dissimilarities [START_REF] Durand | One-t-one correspondences in pyramidal representation: a unified approach[END_REF], and between weakly indexed pyramids and robinsonian dissimilarities [START_REF] Diday | Une représentation visuelle des classes empiétantes : les pyramides[END_REF]. Generalizations and/or unifications of some of these bijections can be found in (3; 11; 6; 18; 7; 2; 12). The present paper focuses on a bijection between semi-proper robinsonian dissimilarities and indexed pre-pyramids, in a general setting where cluster structures are not required to contain all singletons of the ground set, i.e., are not necessarily definite. We give a direct proof of this bijection, showing that the clusters of the indexed pre-pyramid corresponding to a semi-proper robinsonian dissimilarity are particular 2-balls of the considered dissimilarity. This result generalizes its analogue proven by Batbedat (4) in the particular case of definite cluster structures. Moreover, it can be thought of as the third side of a triangular correspondence between indexed pre-pyramids, weakly indexed pyramids and semi-proper robinsonian dissimilarities. Indeed, on the one hand, a bijection between indexed pre-pyramids and weakly indexed pyramids derives as an instance of the general one between indexed cluster structures and weakly indexed closed cluster structures [START_REF] Diatta | One-to-one correspondence between cluster structures and weakly indexed closed cluster structures[END_REF]. On the other hand, the Diday's bijection [START_REF] Diday | Une représentation visuelle des classes empiétantes : les pyramides[END_REF] extends between weakly indexed (not necessarily definite) pyramids and semi-proper robinsonian dissimilarities.

1 Pyramids and Pre-pyramids

Pre-pyramids

Let E be a finite nonempty set. A cluster structure on E is a collection C of subsets of E, satisfying conditions (CS1), (CS2) and (CS2') below: (CS1) the empty set is not a member of C whereas the ground set E is, i.e., ∅ / ∈ C and E ∈ C; (CS2) the set of minimal members of C (w.r.t. set inclusion) partitions E; in other words, these minimal members are non-empty, pairwise disjoint, and they cover E (i.e. their union equals E);

(CS2') every non-minimal member of C is the union of members of C it properly contains, i.e., for all X ∈ C: ∪{Y ∈ C : Y ⊂ X} ∈ {∅, X};

(CS3) there is a linear order, say θ, on E, of which each member C of C is an interval, i.e., for all x, y, z ∈ E: x, y ∈ C and xθzθy imply z ∈ C.

The pair of conditions (CS2) and (CS2') is often replaced by a stronger condition requiring each singleton to be a member of C. Actually, a cluster structure satisfying this strong requirement is said to be total or definite.

A pre-pyramid on E is a cluster structure C onf E, satisfying the condition (CS3) below: (CS3) there is a linear order, say θ, on E, of which each member C of C is an interval, i.e., for all x, y, z ∈ E: x, y ∈ C and xθzθy imply z ∈ C.

The order θ and the collection C are said to be compatible with each other, and subset collections satisfying condition (CS3) are said to admit a compatible order. The condition (CS3) is the specific condition of interval-type subset collections.

Pyramids

To every subset collection can be associated its closure consisting of arbitrary intersections of its members. As we are concerned with collections of nonempty subsets of finite sets, we will consider only finite nonempty intersections. The closure of a subset collection C under (finite) nonempty intersections will be denoted by C, and C will be said to be closed when it satisfies the condition (CS4) below:

(CS4) the intersection of two members of C is either empty or a member of

C, i.e., X, Y ∈ C implies X ∩ Y ∈ C ∪ {∅}.
A pyramid is a closed pre-pyramid. It can be checked that conditions (CS2) and (CS2') are equivalent under (CS1) and (CS4). Pyramidal classification has been introduced by Diday ( 16) and considered or investigated in several works among which we can mention (21; 19).

To show that C is a pyramid when C is a pre-pyramid, it suffices to check that C verifies (CS2). Now this derives from the fact that, by Lemma 1 and Lemma 2 below, every minimal member of C is also a minimal member of C.

Lemma 1 [START_REF] Diatta | One-to-one correspondence between cluster structures and weakly indexed closed cluster structures[END_REF] The conjunction of conditions (CS2) and (CS2') is equivalent to the conjunction of conditions (a) and (e), where:

(a 1 ) C, C ′ ∈ C and C minimal in C imply C ∩ C ′ ∈ {∅, C}; (a 2 ) minimal members of C cover E.
Lemma 2 [START_REF] Diatta | One-to-one correspondence between cluster structures and weakly indexed closed cluster structures[END_REF] The following conditions are equivalent for a collection C of nonempty subsets of E.

(a 1 ) C, C ′ ∈ C and C minimal in C imply C ∩ C ′ ∈ {∅, C}.
(a 3 ) Every minimal member of C is minimal in C.

Indexed pre-pyramids and weakly indexed pyramids

Let C be a cluster structure on E. A pre-index on C is an order preserving map f : (C, ⊆) → (R + , ≤) taking the zero value on minimal members of C, i.e.,

(i) C minimal implies f (C) = 0; (ii) C ⊆ C ′ implies f (C) ≤ f (C ′ ).
In the sequel, we will assume that a pre-index f takes the value zero only on minimal members, hence, f (C) = 0 if and only if C is minimal. A canonical pre-index f c can be obtained by letting f c (C) be the number of elements of the union of members of

C properly contained in C. An index on C is a strict pre-index, i.e., a pre-index f such that C ⊂ C ′ implies f (C) < f (C ′ ). A weak index (6) on C is a pre-index f such that [C ⊂ C ′ and f (C) = f (C ′ )] implies [C = ∩{C ′′ ∈ C : C ⊂ C ′′ }].
When f is a pre-index (resp. an index, a weak index) on a cluster structure C, the pair (C, f ) is said to be pre-indexed (resp. indexed, weakly indexed). Let (C, f ) be a pre-indexed cluster structure on E. Let Inter(C, f ) denote the pair (C, f ), where f is defined on C by

f (C) = min{f (C ′ ) : C ′ ∈ C and C ⊆ C ′ }.
On the other hand, define an f -maximal member of C to be a non-minimal member C ∈ C such that there is no member

C ′ ∈ C such that C ⊂ C ′ and f (C) = f (C ′ ). Let Strict(C, f ) denote the pair (C, f ),
where C is composed of minimal and f -maximal members of C, and f the restriction of f on C. Then Strict(C, f ) is clearly an indexed cluster structure. Moreover, the following result shows that indexed pre-pyramids correspond to weakly indexed pyramids in a one-to-one way. A proof can be found in [START_REF] Diatta | One-to-one correspondence between cluster structures and weakly indexed closed cluster structures[END_REF].

Theorem 1 A robinsonian dissimilarity is a dissimilarity admitting a compatible order, i.e., such that there exists a linear order θ on E such that xθyθz=⇒ max{d(x, y), d(y, z)} ≤ d(x, z).

(i) If (C, f ) is
Robinsonian dissimilarities play an important role in unidimensional scaling problems in archeology [START_REF] Robinson | A Method for Chronologically Ordering Archeological Deposits[END_REF] and in the analysis of DNA sequences [START_REF] Mirkin | Graphs and Genes[END_REF]. See also [START_REF] Warrens | On robinsonian dissimilarities, the consecutive ones property and latent variable models[END_REF] for more references.

Semi-proper robinsonian dissimilarities and indexed pre-pyramids

In this section, we show that semi-proper robinsonian dissimilarities correspond to indexed prepyramids via their associated 2-balls. Let d be a dissimilarity on E. Given x ∈ E and r ≥ 0, the d-ball of center x and radius r is the set B d (x, r) defined as B d (x, r) = {z ∈ E : d(x, z) ≤ r}.

Let now x, y be two (not necessarily distinct) elements of E. Then, the (d, We will use the two following lemmas to prove the bijection between indexed pre-pyramids and semi-proper robinsonian dissimilarities.

∈ C(v, y), d(x, y) ≤ f (C) < f (X), because C ⊂ X, so that d(x, y) < min X∈C(v,y) f (X) = d(v, y).
Assume now that yθv. By an argument of symetry, we can deduce from the previous case that It should be noticed that Theorem 2 still holds and the Diday's bijection [START_REF] Diday | Une représentation visuelle des classes empiétantes : les pyramides[END_REF] can be derived form it, when "B d " and "indexed" are replaced by "B d " and "weakly indexed", respectively [START_REF] Durand | One-t-one correspondences in pyramidal representation: a unified approach[END_REF].

d(x, y) < min X∈C(x,v) f (X) = d(x, v).

2 Correspondences with robinsonian dissimilarities 2 . 1

 21 an indexed cluster structure on E, then Inter(C, f ) is a weakly indexed closed cluster structure on E. Moreover, Strict(Inter(C, f )) = (C, f ). (ii) Conversely, if (C, f ) is a weakly indexed closed cluster structure on E, then Strict(C, f ) is an indexed cluster structure on E. Moreover, Inter(Strict(C, f )) = (C, f ). Robinsonian dissimilarities Let us recall that a dissimilarity on E is a map d : E × E → R + satisfying d(x, x) = 0 and d(x, y) = d(y, x). It is said to be semi-proper if d(x, y) = 0 implies d(x, z) ≤ d(y, z) for all z.

  2)-ball (or simply 2-ball) generated by the pair x, y is the set B d xy defined as B d xy = B d (x, d(x, y)) ∩ B d (y, d(x, y)). It may be noted that B d xy = B d (x, d(x, x)) when x = y. Let diam d be the function defined on nonempty subsets X of E by diam d (X) = max{d(x, y) : x, y ∈ X} and let B d be the set defined by B d = {B d xy : x, y ∈ E and diam d (B d xy ) = d(x, y)}.

Proof.

  Let d be a a semi-proper robinsonian dissimilarity on E. Clearly, E ∈ B d since E = B d xy with x, y such that d(x, y) = diam d (E). Moreover, as d is semi-proper, the 2-balls of the form B d xx are the minimal members of B d . Hence they cover E, and, for all x, y, z, B d xx ∩ B d yz ∈ {∅, B d xx }. Finally, by Lemma 3, the elements of B d are intervals of any order compatible with d, proving that B d is a pre-pyramid on E. On the other hand, it is a clear fact that, for B ∈ B d , diam d (B) = 0 if and only if B = B d xx for some x ∈ E, as d is semi-proper, i.e., if and only if B is minimal. Moreover, it follows obviously from Lemma 4 that diam d is an index on B d , proving the direct assertion. Conversely, let (C, f ) be an indexed pre-pyramid on E. For all x, y ∈ E, denote by C(x, y) the collection of members of C that contain x and y, i.e. C(x, y) = {C ∈ C : x, y ∈ C}. Let d be the dissimilarity on E defined by d(x, y) = min{f (C) : C ∈ C(x, y)}. The dissimilarity d is robinsonian. Let xθyθz, where θ is an order compatible with C. Then every member of C containing x and z contains also y. Therefore, max{d(x, y), d(y, z)} = max{ min C∈C(x,y) f (C), min C∈C(y,z) f (C)} ≤ min C∈C(x,z) f (C) = d(x, z), as required. The dissimilarity d is semi-proper. Indeed, let x, y ∈ E such that d(x, y) = 0. Then there is a member of C containing x, y, say C xy , such that f (C xy ) = 0. On the other hand, by condition (CS2), there is a minimal member of C containing x, say C x . Thus C xy = C x since f (C x ) = 0 = f (C xy ) and f is an index. Let z ∈ E. Now C xy being minimal, for all C ∈ C(x, z) we have, by Lemma 1-(a 1 ), C xy ⊆ C. Therefore, every member of C containing x, z contains also y, z, so that d(y, z) = min C∈C(y,z) f (C) ≤ min C∈C(x,z) f (C) = d(x, z). The pairs (B d , diam d ) and (C, f ) are equal. Let B := B d xy ∈ B d . By Lemma 4, we assume, w.l.g., its bounds be x and y. Let A xy ∈ Argmin{f (C) : C ∈ C(x, y)}. Then B ⊆ A xy since A xy is an interval of θ. Let us prove the reverse inclusion. Denoting u and v the bounds of A xy , we have d(u, v) ≤ f (A xy ) = d(x, y). Now, as x, y ∈ A xy we get d(x, y) ≤ d(u, v) = diam(A xy ). Then, by compatibility of d with θ, we have max{d(x, u), d(x, v), d(y, u), d(y, v)} ≤ d(u, v) = d(x, y), i.e., u, v ∈ B, proving that B = A xy , hence B d ⊆ C. To prove the reverse inclusion, let C ∈ C with bounds x and y (xθy). Then, as B d xy is an interval of θ, C ⊆ B d xy . In order to prove the reverse inclusion, let v ∈ C = [x, y]. Let us first consider the case where vθx. Then for all X

  Thus v / B d xy , proving that B d xy ⊆ C. Therefore, C = B d xy and, by the way, x and y are the bounds of B d xy . Hence C ∈ B d , proving that B d= C. In addition every C(x, y) contains C, so that f (C) = d(x, y) = diam d (B d xy ), proving that (B d , diam d ) = (C, f ). The dissimilarity d is unique. Let d ′ be a semi-proper robinsonian dissimilarity such that (B d ′ , diam d ′ ) = (C, f ). Let x, y ∈ E with xθy. Let u be the smallest element of E (w.r.t. θ) such that d ′ (u, y) = d ′ (x, y) and let v be the greatest element of E such that d ′ (u, v) = d ′ (u, y) (uθx and yθv). Then d ′ (u, v) = d ′ (x, y) and B d ′ uv = [u, v] ∈ B d ′ = C = B d . Thus, as x, y ∈ B d ′uv (since uθxθyθv and B d ′ uv is an interval containing u and v),d(x, y) ≤ f (B d ′ uv ) = diam d ′ (B d ′ uv ) = d ′ (u, v) = d ′ (x, y).Assume now that there exists a member X ∈ C(x, y) such that f (X) < d ′ (x, y). If w and z are the bounds of X with wθz, then wθxθyθz.But X ∈ C = B d ′ implies X = B d ′ wz , so that f (X) = diam d ′ (B d ′ wz ) = d ′ (w, z). It follows then that d ′ (w, z) < d ′ (x,y), which contradicts the compatibility of d ′ with θ. Hence d ′ (x, y) ≤ min C∈C(x,y) f (C) = d(x, y), proving that d ′ (x, y) = d(x, y). Therefore d ′ = d, as required.


Susan M Browne
email: susan.browne@ucd.ie

Mohamed Al-Rubeai
email: m.al-rubeai@ucd.ie

Defining viability in mammalian cell cultures

Keywords: Apoptosis, Biopharmaceuticals, Mammalian cell culture, Necrosis, Viability

A large number of assays are available to monitor viability in mammalian cell cultures with most defining loss of viability as a loss of plasma membrane integrity, a characteristic of necrotic cell death. However, the majority of cultured cells die by apoptosis and early apoptotic cells, although non-viable, maintain an intact plasma membrane and are thus ignored. Here we measure the viability of cultures of a number of common mammalian cell lines by assays that measure membrane integrity (a measure of necrotic cell death) and assays that measure apoptotic cells, and show that discrepancies in the measurement of culture viability have a significant impact on the calculation of cell culture parameters and lead to skewed experimental data.

Introduction

In mammalian cell cultures producing protein therapeutics optimum viability is essential in extending the duration of productive culture and maintaining product quality. Enzymes and incompletely processed product released from lysed cells can degrade protein product, alter glycosylation profiles and affect downstream purification [START_REF] Gramer | Glycosidase activities in Chinese-hamster ovary cell lysate and cell-culture supernatant[END_REF][START_REF] Goldman | Monitoring proteolysis of recombinant human interferon-gamma during batch culture of Chinese hamster ovary cells[END_REF][START_REF] Hansen | Proteolytic cleavage of recombinant two-chain factor VIII during cell culture production is mediated by protease(s) from lysed cells -The use of pulse labelling directly in production medium[END_REF]. Non-viable cells in culture also have an inhibitory effect on the productivity of the remaining viable population, with a recent study in a hybridoma line showing an inverse correlation between the proportion of apoptotic cells at the beginning of culture and subsequent antibody production [START_REF] Gregory | Inhibitory effects of persistent apoptotic cells on monoclonal antibody production in vitro. Simple removal of non-viable cells improves antibody productivity by hybridoma cells in culture[END_REF]). Thus, careful monitoring and maintenance of viability in culture is essential.

For the majority of common protocols viability is defined by an intact plasma membranemeasured either through dye exclusion or the release of intracellular enzymes such as lactate dehydrogenase. Other assays using the dyes MTT or Alamar Blue measure metabolic activity.

However, these criteria are only relevant to necrotic cell death, whereas the majority of cells in culture die via apoptosis [START_REF] Singh | Cell death in bioreactors -a role for apoptosis[END_REF]. Unlike necrosis, which is characterised by a sudden loss of membrane integrity and leakage of intracellular contents, apoptosis is a tightly regulated process of cellular disassembly. Early apoptotic cells, although fully committed to the cell death program, are metabolically active and have an intact plasma membrane, with membrane integrity maintained until the late apoptotic/secondary necrotic phase. However, this fraction of nonviable cells is often overlooked. In this study we compare cell culture data that has been calculated based on the viability values obtained from two different assaysmeasuring either necrotic or apoptotic cellsto highlight the effect that different definitions of viability can have on experimental data.

Materials and methods

Cell lines and culture conditions GS (glutamine synthetase)-NS06A1 (100) 3 cells were cultured in DMEM/F12 without glutamine containing 10% (v/v) FBS, 2% GS Supplement and 30 µM MSX. The NS1 derived murine hybridoma (TB/C3) was maintained in RPMI1640 containing 5% (v/v) FBS. CHOK1 cells were maintained in DMEM/F12 containing 5% (v/v) FBS and 2 mM L-glutamine. The hepatocyte cell line, HepZ, and HEK293 cells were maintained in DMEM/F12 containing 10% (v/v) FBS and 2 mM L-glutamine.

Measurement of cell number, viability and apoptosis

Viable cell number and viability were measured by Trypan Blue exclusion or by propidium iodide (PI) staining (1 µg/ml final concentration).

Apoptotic fractions were determined by the annexin-V-FITC/PI assay. Briefly cells were washed in PBS and resuspended in annexin binding buffer (10 mM HEPES, 140 mM NaCl, 2.5 mM CaCl 2 , pH 7.4). Annexin V-FITC reagent (BD Biosciences) was added and samples incubated for 20 min in the dark. PI was added to give 1 µg/ml and samples were analysed by flow cytometry.

Measurement of apoptosis by fluorescent microscopy was carried out as previously described [START_REF] Simpson | Prevention of hybridoma cell death by bcl-2 during suboptimal culture conditions[END_REF]. Briefly cell suspensions were mixed with Acridine Orange (AO)/PI, each at 10 µg/ml, followed by fluorescent microscopy.

Monoclonal antibody measurement

Recombinant cB72.3 antibody in supernatant samples was measured by ELISA as previously described (Tey and Al Rubeai 2004).

Results and discussion

The apoptotic profile for a typical batch culture of a GS-NS0 myeloma cell line as measured by the annexin-V-FITC/PI assay is shown in Figure 1. This assay is based on binding of annexin-V to phosphatidyl-serine (PS), which is characteristically translocated to the outer plasma membrane of the cell in the early phase of apoptosis, and uptake of the dye PI upon loss of membrane integrity (Vermes et al. 1995). Using these parameters the culture can be divided into fractions of viable (annexin-V negative/PI negative) cells, early apoptotic (annexin-V positive/PI negative) cells that have entered apoptosis but have not yet lost membrane integrity, and late apoptotic/secondary necrotic (annexin-V positive/PI positive) cells where PS is exposed and membrane integrity has been lost. Based on this assay a drop in culture viability relating to the onset of death phase can be seen from 72 h (Figure 1A), with a corresponding increase in the early apoptotic population (Figure 1B); however, there is no increase in late apoptotic cells, relating to loss of membrane integrity, until 120 h (Figure 1C). Here the early apoptotic population represents a significant proportion of non-viable cells particularly at the early stages of death phase. At 96 and 120 h, early apoptotic cells account for 15% and 34% of the total cells in culture, respectively, representing more than 55% of the dead cell population at both timepoints. Based on many of the assays generally used for viability measurements, however, this population of cells would be overlooked and the actual percentage viability of the culture would be significantly overestimated.

To show the extent of this effect we have compared data based on measurement of viable cells by the annexin-V/PI assay to that based on membrane integrity alone through exclusion of PI.

The difference in percentage viability as determined by the two assays ranges from just under 20% in the early stages of culture to a peak of 48% at 120 h (Figure 2A). Calculating the viable cell density of the culture based on these different viability measurements leads to a significant difference in the viable growth profile (Figure 2B). The calculation of other cell culture parameters is also affected. Although there is no major impact on the calculation of the doubling time of the culture, there are marked differences in the integral of viable cell concentration (IVCC), which was overestimated by the membrane integrity assay, and of specific production of monoclonal antibody cB72.3, which was actually underestimated based on this assay (Table 1, The impact of these differences in determination of viability can be seen to a greater or lesser extent in different cell lines depending on the duration of a particular cell line's transition through the early phase of apoptosis. Figure 3 shows a comparison of cell number and culture viability for a range of common cell lines where viability, as determined by membrane integrity alone, was measured by Trypan Blue exclusion and viability based on the non-apoptotic cell fraction was measured by AO/PI staining and fluorescent microscopy where early apoptotic cells can be identified by visualisation of condensed chromatin stained by AO. Based on these assays the differences in viability measured in early death phase (where the discrepancies between assays is most pronounced) were similar for HEK293, HepZ (a hepatocyte cell line), and TB/C3 (a hybridoma cell line) cell lines at approx. 15%, for CHO-K1 however, a difference of 62% was measured (Figure 3).

In conclusion, these results show that an accurate measurement of culture viability can only be established through the use of an assay that takes into account all non-viable fractions of the cell populationearly apoptotic, late apoptotic and necrotic. The membrane integrity based assays that are widely used cannot provide a true measure of the level of viability of a culture leading to skewed results and bringing into question the accuracy of experiments. A thorough discussion is required as to what constitutes a "viable" cell and a standardised definition of viability needs to be put in place.

Tey BT, Al Rubeai M (2004)

Figure

  Figure 2 C&D).

Figure Captions Figure 1 -

 Captions1 Figure Captions

Figure 3

 3 Figure 3 -(A) Viable cell concentration and (B) viability as measured by trypan blue exclusion

Table 1 -Comparison of cell culture calculations for the NS06A1 cell line based on culture viability as defined by loss of membrane integrity (PI only) or PS exposure (annexin/PI). Results are presented as mean value of 3 replicates ± Standard deviation.

 1

	PI	ANNEXIN/PI	DIFFERENCE


Stepan Boitsov

Vera Petrova

Henning K B Jensen

Anna Kursheva

Ivan Litvinenko

Yifeng Chen

Jarle Klungsøyr

Petroleum-related hydrocarbons in deep and subsurface sediments from South-Western Barents Sea

Keywords: Biomarkers, hopanes, sediments, pockmarks, PAH, alkanes, Barents Sea

Subsurface sediments from a pockmark area in South-Western Barents Sea have been earlier found to contain elevated levels of petroleum-related polycyclic aromatic hydrocarbons. This work describes a comprehensive analysis of various biomarkers, including the highly sourcespecific hopanes, in a 4.5 m long gravity core from the same area, together with subsurface sediment samples from other areas in the region without pockmarks present ("background samples"). A clear difference between the pockmark gravity core and the background sediment cores was found, both with regard to genesis and the level of transformation of organic matter. A number of indicator parameters, such as methylphenanthrene index (MPI-1), point towards a significantly higher maturity of hydrocarbons in the pockmark core throughout its length as compared to the other sampled locations. Higher contents of microbial hopanoids (hopenes) may indicate the former presence of petroleum. These findings confirm the hypothesis of a natural hydrocarbon source in the deeper strata present in the studied location with pockmarks.

Introduction

The Barents Sea continental shelf is rich in oil and gas resources. In the Norwegian sector, it may contain 1199 million standard cubic meters of oil equivalents (Sm 3 o.e.) according to the latest estimates made by Norwegian Oil Directorate [START_REF] Npd | [END_REF]. Most of this, 910 million Sm 3 o.e., are undiscovered resources (ibid.) The ongoing MAREANO program, a long-term, multi-institutional Norwegian project aiming at extensive biological, geological and 1 To whom the correspondence is to be addressed. Address: Institute of Marine Research, P.B. 1870 Nordnes, N-5817 Bergen, Norway. Tel.: +47-55236394, Fax: +47-55238555, email: stepan.boitsov@imr.no Barents Sea [START_REF] Mareano | Available on the internet at www[END_REF][START_REF] Boitsov | Natural background and anthropogenic inputs of polycyclic aromatic hydrocarbons (PAH) in sediments of South-Western Barents Sea[END_REF][START_REF] Chand | Pockmark-like depressions near the Goliat hydrocarbon field, Barents Sea: Morphology and genesis[END_REF]. The area of Ingøydjupet, located some 80 km North-West of the coast of Norway, was found to contain a pockmark field (depressions approximately 40-60 m in diameter and 6 m deep [START_REF] Chand | Pockmark-like depressions near the Goliat hydrocarbon field, Barents Sea: Morphology and genesis[END_REF]). Pockmarks are geological formations which may signify former or recent seepages of hydrocarbon gas or fluids from formations below the sediment surface (e.g. [START_REF] Hovland | Seabed pockmarks and seepages. Impact on biology, geology and the environment[END_REF]. Levels of predominantly petrogenic polycyclic aromatic hydrocarbons (PAH) were found to increase in subsurface sediment layers (below 15 cm depth), also when corrected for total organic carbon (TOC) levels [START_REF] Boitsov | Natural background and anthropogenic inputs of polycyclic aromatic hydrocarbons (PAH) in sediments of South-Western Barents Sea[END_REF]. A local natural petroleum source was suggested as a possible explanation (ibid.) This is not surprising in view of the proximity of the Goliat oil field (see Fig. 2). According to [START_REF] Juntilla | Late Glacial -Holocene clay minerals elucidating glacial history in the South-Western Barents Sea[END_REF], in other gravity cores of similar lengths taken within the area both Holocene and glaciomarine sediments are encountered.

A more detailed geochemical study was carried out to confirm the dominating source of hydrocarbons in sediments. A number of parameters have been used for this purpose, based either on the levels of source-specific compounds (e.g. a biogenic source in case of elevated levels of certain PAH compounds as perylene, retene, cadalene) or on the ratios and other relations between specific compounds [START_REF] Venkatesan | Organic geocemistry of marine sediments in Antarctic region: marine lipids in McMurdo Sound[END_REF][START_REF] Neff | Bioaccumulation in marine organisms[END_REF][START_REF] Peters | The biomarker guide. Biomarkers and isotopes in the environment and human history[END_REF].

One such useful ratio is methylphenanthrene index, MPI-1, providing an indication of the petrogenic input in the PAH levels. It is calculated according to the following formula: MPI-1=1.89(2-MP+3-MP)/[P+1.26(1-MP+9-MP)] where P is phenanthrene and MP is methylphenanthrene [START_REF] Peters | The biomarker guide. Biomarkers and isotopes in the environment and human history[END_REF].

Aliphatic hydrocarbons (n-alkanes) may also serve as indicators of both genesis and thermal maturity of organic matter [START_REF] Peters | The biomarker guide. Biomarkers and isotopes in the environment and human history[END_REF]. These compounds tend to be less specific than PAH but several ratios can be useful, such as odd-to-even predominance index (OEP) and pristane/phytane ratio (summarised by [START_REF] Commendatore | Hydrocarbons in Coastal Sediments of Patagonia, Argentina: Levels and Probable Sources[END_REF]. OEP is used to determine maturity of organic matter, as odd-carbon-numbered alkanes dominate immature source rocks (OEP>>1 or OEP<<1)), while mature rocks have little or no carbon-number preference (OEP≅1) [START_REF] Peters | The biomarker guide. Biomarkers and isotopes in the environment and human history[END_REF]. A high pristane/phytane ratio also indicates high thermal maturity and may further be used to determine redox conditions in the source rock, Pr/Ph<1 indicating anoxic and Pr/Ph>1 indicating oxic conditions [START_REF] Peters | The biomarker guide. Biomarkers and isotopes in the environment and human history[END_REF]). An additional

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parameter is isoprenoid/n-alkane ratio, which may be expressed as K iso =(Pr+Ph)/(C 17 +C 18 ), which decreases with thermal maturity as more n-alkanes are generated from kerogen [START_REF] Peters | The biomarker guide. Biomarkers and isotopes in the environment and human history[END_REF].

Another, somewhat more reliable way of confirming petrogenic origin of hydrocarbons is to analyse geochemical biomarkers specific for petroleum, such as hopanes, in the same samples. Hopanes are aliphatic pentacyclic triterpanes formed in diagenetic processes and abundant in oil. Precursors of hopanes, bacteriohopanols, are a part of bacterial membranes, which is the reason for the ubiquitous nature of hopanoid-type compounds. They are found in all sediments and have been referred to as the most abundant natural products on Earth [START_REF] Ourisson | Hopanoids. 1. Geohopanoids: the most abundant natural products on Earth?[END_REF]. Hopanes are strongly resistant to biodegradation and to weathering otherwise, and are therefore definitive markers of the presence of oil, former or current [START_REF] Ourisson | Hopanoids. 1. Geohopanoids: the most abundant natural products on Earth?[END_REF][START_REF] Volkman | Identification of natural, anthropogenic and petroleum-hydrocarbons in aquatic sediments[END_REF][START_REF] Prince | 17α(H),21β(H)-Hopane as a conserved internal marker for estimating the biodegradation of crude oil[END_REF][START_REF] Peters | The biomarker guide. Biomarkers and isotopes in the environment and human history[END_REF]. For the same reason, they have been used as "natural internal standards" to measure the degree of biodegradation [START_REF] Prince | 17α(H),21β(H)-Hopane as a conserved internal marker for estimating the biodegradation of crude oil[END_REF]. Stereoisomers of biological origin are maturity-sensitive, degrading under thermal and pressure influence. Various maturity parameters exist based on the relative abundances of two stereoisomers. One of them is T s /T m ratio ("terpane stable to terpane maturable", see Fig. 1), while another one is the ratio of the biogenic precursor of these two terpanes, T e (Fig. 1), to their sum, T e /(T s +T m ). These ratios may provide information on the degree of transformation of organic matter [START_REF] Seifert | Applications of steranes, terpanes and monoaromatics to maturation, migration and source of crude oils[END_REF][START_REF] Farrimond | Biomarker maturity parameters: the role of generation and thermal degradation[END_REF]. Another highly specific indicator of immature to early oil generation is 22S/(22S+22R) isomerisation of homohopanes as C 31 , giving characteristic doublets in m/z 191 mass chromatograms (hopanograms) [START_REF] Peters | The biomarker guide. Biomarkers and isotopes in the environment and human history[END_REF].

Similarly, the ratios of hopane С 30 isomers, (αβ/(αβ+βα) C 30 and ββ/(αβ+βα) С 30 , provide information on thermal maturity as ββ-hopanes (biohopanes) convert readily to αβand βαconfigurations, and their high levels indicate low degree of maturity contrary to αβ-hopanes [START_REF] Peters | The biomarker guide. Biomarkers and isotopes in the environment and human history[END_REF]. A biomarker fingerprint (e.g. a hopanogram) may yield information on the particular source and has been used to determine specific oils (e.g. [START_REF] Boehm | Resolving the origin of thee petrogenic hydrocarbon background in Prince William Sound, Alaska[END_REF][START_REF] Bartolomé | Chemical fingerprinting of petroleum biomarkers in biota samples using retention-time locking chromatography and multivariate analysis[END_REF][START_REF] Saha | Sources of sedimentary PAHs in tropical Asian waters: Differentiation between pyrogenic and petrogenic sources by alkyl homolog abundance[END_REF]. Due to a large number of isomers and low biomarker levels in more recent sediments, GC-MS analysis of biomarkers requires a finetuned analytical method and a thorough sample purification procedure. Several methods have been described [START_REF] Boonyatumanond | Distribution and origins of polycyclic aromatic hydrocarbons (PAHs) in riverine, estuarine and marine sediments in Thailand[END_REF]Zakaria et al., 2001). Other less specific biomarkers, such as alkanes and PAH described above, may provide a confirmation of the

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conclusions drawn from hopane analysis, and a combined analysis of various types of biomarkers has been used to create an integrated approach to the study of the origin of hydrocarbons in sediments (e.g. [START_REF] Yunker | Terrestrial and marine biomarkers in a seasonally ice-covered Arctic estuary -integration of multivariate and biomarker approaches[END_REF].

Figure 1. Chemical structures of 22,29,22,29,and 22,29,-ene (Te).

Methods

Sampling (UiT-NGU, IMR).

This taken in the same area in the same year were comprised of between 86 and 95 % silt and clay [START_REF] Jensen | Mareano 2006 -Environmental Geochemistry Results from Tromsoflaket, Ingoydjupet, Lopphavet and Soroysundet[END_REF]. The gravity core was frozen whole onboard and taken to the laboratory for analyses. Prior to analyses, it was sliced in 2 cm-thick samples, which were subsequently air-dried. A detailed description of the samples taken to geochemical analyses is given in 1). A multicorer was used to collect the samples at stations R406 and R488, whereas a boxcorer was used at station R422. The cores were between 30 and 42 cm long, lithologically homogenous, with low carbonate contents [START_REF] Jensen | Mareano 2009 -Environmental Geochemistry Results from Eggakanten[END_REF][START_REF] Jensen | Mareano 2009 -Environmental Geochemistry Results from Eggakanten[END_REF]. The cores were sliced onboard into 1 cm slices and kept frozen until the analyses. Prior to analyses, the samples were air-dried. A detailed description of the samples taken for geochemical analyses is given in Table 1, and the locations of the sampling stations are shown in Fig. 2. One set of sediment sub-samples was analysed for weight percentages (wt. %) of total organic carbon (TOC). For the TOC analyses, aliquots (~200 mg) of the samples were treated with 10 % (volume) hydrochloric acid (HCl) at 60ºC to remove carbonate, and then washed with distilled water to remove HCl. We caution the reader that the possible loss of organic material by acid leaching is not taken into account. The samples were dried overnight (50ºC) and then analysed using a LECO CS 244 analyser.

Grain size analyses of total sediment were obtained from laser diffraction techniques (Coulter LS 2000) (for details, see [START_REF] Xu | Characterization: Light Scattering Methods[END_REF], and references therein) and wet sieving. The Coulter LS 2000 measured the fractions from 0.4 µm to 2 mm while particles larger than 2 mm were determined by wet sieving. Prior to each analysis, the samples were freeze-dried. The grain size distribution is determined with respect to percent volume, with the assumption of uniform density of the sample.

Sample pre-treatment (extraction and cleanup, IMR)

Air-dried samples were weighed (approximate weights are given in Table 1) and loaded in ASE (Acclerated Solvent Extractor) cells together with diatomaceous earth. Internal standard, isoadiantane-d 2 (100 ng, 100 µl hexane solution), was added to the mixture in the cells.

Conditions of ASE extraction on Dionex ASE300 apparatus were as follows: 2 cycles, 60% flush volume, 150°C. Solvent used was 1:1 mixture of hexane and dichloromethane. Solvent volume was reduced to 0.5 ml on a rotavap (room temperature) and copper added for sulphur removal overnight. After further volume reduction, samples were cleaned-up on Bond Elute

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silica SPE columns (3 cc, 500 mg), eluted with hexane. Final sample volume was 300 µl hexane solution.

Sample pre-treatment (sulphur removal and fractionation, VNIIO)

Sample extracts from the gravity core still contained traces of sulphur after sample pretreatment. These were therefore further cleaned-up on an activated copper column to remove remaining sulphur. The solvent was removed by a rotavap (Т<40ºC). The samples were then fractionated on silica gel to extract the methane-naphthenic and aromatic hydrocarbon fractions.

GC-MS analysis (VNIIO)

Hydrocarbon Alkanes, hopanes and steranes were analysed on the following system: GC capillary column HP DB 30m x 0.25mm x 0.25 µm, stationary phase 5%phenyl-95%methylpolysiloxane.

Carrier gas was helium, flow rate 1,2 ml/min; injector temperature 320 ºС; oven temperature 152, 154, 178, 192, 202, 206, 228, 252, 276 and 278. Alkylated PAH were analysed by selective ion masses m/z 142, 156, 192, 206, 220, 216, 234 and 242.

Results and Discussion

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TOC and saturated hydrocarbons.

The distribution of total organic carbon (TOC) in the gravity core from station 06JM-12 in Ingøydjupet is shown in Fig. 3a and ranges from 0.4 to 0.8 % throughout the core. The content and distribution of total sulphur (TS) in the core is probably caused by the change in diagenetic conditions in the sediments, from oxidative conditions in the surface layer (8-47 cm) to reducing conditions (below 55 cm), see Fig. 3a. The average contents of saturated hydrocarbons in the core from station 06JM-12 (Fig. 3b), 1600 ng/g sediment, does not exceed the typical values of similar lithological varieties of the West-Arctic continental shelf (Yunker et al., 1996;[START_REF] Dahle | Polycyclic aromatic hydrocarbons (PAHs) in Norwegian and Russian Arctic marine sediments: concentrations, geographical distribution and sources[END_REF]. A bimodal character of alkane distribution (Fig. 4) indicates a mixed sapropel-humus composition of the original organic matter (C 14-C 19 /C 14 -C 35 = 0.3 average value). Odd-to-even predominance index (OEP), which reflects the level of transformation of the humus part of organic matter (OM), corresponds to the diagenetic stage of the transformation (ОЕР 27-31 =3.0). In the case of low molecular weight n-alkanes (С 15 -С 19 ), ОЕР does not exceed 1, which may be due to their selective degradation during early diagenesis [START_REF] Grimalt | Sources and occurrence of C-C n-alkane distribution with carbon-number preference in sedimentary environments[END_REF][START_REF] Venkatesan | The lipid geochemistry of Antarctic marine sediments: Bransfild strait[END_REF] Figure 4. N-alkane distribution in the gravity core from station 06JM-012. Numbers for colored lines to the right are the sampled depths in cm.

At the same time, in three of the studied intervals of the core (20 -22 cm, 135 -137 cm, 222 -224 cm), n-alkane distributions are anomalous. Absolute contents of saturated hydrocarbons in these intervals increase 2-to 3-fold as compared to the rest of the samples (Fig. 3b). When corrected for TOC contents, the increase is 3-to 6-fold (Fig. 3c). The distribution of n-alkanes (Fig. 4) is also anomalous, with low molecular weight compounds (C 14 -C 19 /C 14 -C 35 = 0.8) dominating, with a maximum at n-C 16 .

The observed peculiarities in n-alkane distribution seem to indicate a pulsed influx of low molecular weight hydrocarbons (C 14 -C 19 ) to the sediments. This may be indicative of an abrupt change in sedimentation conditions during a marine transgression and/or abrupt

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increase in primary bioproductivity in the marine environment since these compounds are markers of lipids of biotic origin [START_REF] Venkatesan | The lipid geochemistry of Antarctic marine sediments: Bransfild strait[END_REF][START_REF] Grimalt | Sources and occurrence of C-C n-alkane distribution with carbon-number preference in sedimentary environments[END_REF][START_REF] Petrova | Geochemistry of organic matter of bottom sediments in the rises of the central Arctic Ocean[END_REF]. However, this possibility does not seem plausible in view of the lithological homogeneity of the sediment core and of the increase of anomalies when the data are normalised to TOC. Alternatively, the increase in low molecular weight n-alkanes'

contents may indicate an input of petrogenic hydrocarbons. This fits well with the earlier geochemical studies of the sediments from this region [START_REF] Jensen | Mareano 2006 -Environmental Geochemistry Results from Tromsoflaket, Ingoydjupet, Lopphavet and Soroysundet[END_REF], according to which there is indicated a diffuse fluid flux of hydrocarbons from the bedrock. This facilitates the formation of a wide pockmark area in the zone of unconsolidated fine-grained postglacial deposits. The influence of deep fluids on the composition of OM/hydrocarbons seems quite possible since this increase parallels a strong decrease in the hydrocarbons' odd-to-even predominance index (ОЕР С 17 -C 19 < 1) (Fig. 5a), which is typical for petroleum n-alkanes.

The distribution of alkane markers of terrestrial vegetation (С 27 -C 31 , Fig. 5b) experiences no significant changes in the sediment core, and the value of ОЕР>2,5 corresponds to an insignificant (diagenetic) degree of transformation. The type of distribution and the ratios of isoprenoids (Pr/Ph, Kiso=(Pr+Ph)/(C 17 +C 18 ), Fig. 5c) seem to be determined by the background and are weakly influenced by the petrogenic constituent. Sediment samples from the pockmark area, originating from the same sea depth (420-440 m)

and core intervals (9-46 cm) as those from background areas, are in general similar in their hydrocarbon composition (Fig. 6b) to the background samples. The origin of organic matter is therefore also similar, a sapropel-humus one (С 14 -C 19 /С 20 -C 35 = 0.35, ОЕР 27 = 2.9). An Correspondingly, an enrichment/contribution of OM with components of terrigenous origin seen in Fig. 7a (humus OM) tied into persistent organomineral complexes within the sediments takes place .

Figure 7. N-alkane distribution in the sediments from the continental slope and its foot.

Numbers for colored lines to the right are the sampled depths in cm.

The nature of the observed anomaly was further studied by means of GC-MS analysis of the non-specific groups of molecular markers. It is known that homological series of alkylsubstituted cyclanes, e.g. n-alkylcyclohexanes, are a characteristic petroleum component [START_REF] Kashirtsev | Organic geochemistry of oils in the east of the Siberian Platform[END_REF]. At the same time, their presence is unlikely in Holocene-Quaternary deposition in the marine environment. A comparison of mass-fragmentograms (Fig. 8) by the characteristic fragment ion (m/z 83) has shown that n-alkylcyclohexanes are absent in the background samples. In the samples from the pockmark area, on the other hand, it was the same intervals that exhibited anomalies in n-alkane distribution, which were also enriched in n-alkylcyclohexanes.

Figure 8. Mass-fragmentograms of n-alkylcyclohexanes distributions in various samples.

Cyclic hydrocarbons.

Cyclic hydrocarbons are represented in the gravity core 06 JM-012 mostly by triterpanes. An interpretation of the distribution and composition of steranes in the samples was impossible due to their low contents. However, a prevalence of hopanes over steranes may indicate a

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significant role of microbiota in the formation of bacterial OM within the sediments [START_REF] Peters | The biomarker guide. Biomarkers and isotopes in the environment and human history[END_REF][START_REF] Greenwood | Bacterial biomarkers thermally released from dissolved organic matter[END_REF] Triterpanes in the sediments from the pockmark area are represented by a wide spectrum of compounds (Fig. 9). Comparing to the similar facies and intervals of the cores from background areas, hopenes and ββ-hopanes play a more significant role in these samples, which is typical for weakly transformed OM. These results are not contradictory, since the composition of triterpanes in the studied sediment core may be formed by various sources, including re-deposited, deeply transformed material of continental source rocks.

Figure 10. Triterpane distribution in the sediment core from station 06JM-12.

The general trend in the change of the hopane composition towards deeper layers of the core reflects a decrease in the level of thermal maturity of hydrocarbons and of organic matter in general (Fig. 10). This follows from the reverse trends in the ratios of hopane С 30 epimers, (αβ/(αβ+βα) C 30 , ββ/(αβ+βα) С 30 ). The contents of biohopane (ββ) increase towards deeper layers of the core. This may be due to the decreasing role of mature re-deposited source rocks in sedimentation processes or as a result of petroleum hydrocarbons having migrated into specific intervals with higher permeability than the adjacent denser sediments. Besides, in situ diagenetic processes should also lead to an increase in the contents of transformation products of biogenic precursors of hopanes, hopanols (via hopenes and biohopanes). This fits well to the character of relative hopene distribution in general, and that of the specific hopene, С 27 -22,29,30-trisnorhop-17(21)-ene (Te, see Fig. 1), in the group of trisnorhopanes, for which it is the biogenic precursor (Fig. 10). This does not exclude the possibility of petrogenic hydrocarbons influx to the sediments. On the contrary, an increase in the levels of microbial life products in the sediments (in particular, biohopanols and therefore hopenes) may be due to the presence of an extra food source for hydrocarbon-oxidating bacterial

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communities [START_REF] Innes | Early diagenesis of bacteriohopanoids in recent sediments of Lake Pollen, Norway[END_REF]. This suggestion is indirectly confirmed by the similar trends in distributions of n-alkanes (Fig. 5a) and hopenes (Fig. 10).

On the other hand, the surface sediments from the pockmark area are enriched in geohopanes (αβ-and βα-hopanes) of considerable thermal maturity ((22S/22S+22R) C 31 = 0,70). This may be due to ancient pre-Quaternary source rock contributing to the sediments due to erosion (Yunker et al., 1996) or the migration of thermally mature petroleum hydrocarbons into the surface sediments. Their input into the surface Quaternary sediments may occur both by means of sub-aquatic or continental erosion and by means of migration of deep fluids, as for example leading to venting through the sediment surface and the formation of mud volcanoes [START_REF] Lein | Chemical and isotopic evidence for the nature of fluid in CH4-containing sediments of the Haakon Mosby mud Volcano[END_REF].

Polycyclic aromatic hydrocarbons (PAH).

The character of PAH distribution in the core from station 06JM-12 indicates the dominant role of natural (biogenic and/or petrogenic) sources. Thus, the Fl/202 ratio, varying weakly along the core, has the average value of 0.38 and does not exceed 0.46, which means an insignificant contribution of pyrogenic components (Fig. 11) (Yunker et al, 2002).

The distribution of such typical aromatic biomarkers as retene, cadalene and perylene (Fig. 11), genetically related to terrestrial higher plants, clearly shows the trend in the processes of transformation of original organic matter under reducing diagenetic conditions. The increase of the relative (Per/∑PAH) and absolute (ng/g ТОС) contents of these compounds in the depth of the core is due to their diagenetic formation, by means of transformation of their biogenic precursors such as quinine pigments, di-and sesquiterpenoids. (Bouloubassi and Saliot,1993;[START_REF] Peters | The biomarker guide. Biomarkers and isotopes in the environment and human history[END_REF] Unlike the trends found for biogenic PAH, the relative increase in the amounts of petrogenic PAH (such as phenanthrene and its alkylated homologues) is observed in the upper part of the core (Fig. 11), reaching more than 50% of total PAH levels. This may be due to a more intensive input of hydrocarbon source rocks to the sediments. However, it may also be possible that the input and selective accumulation of low-molecular weight PAH occurs due to a vertical migration of hydrocarbons as part of fluids from deeper strata, typical for oiland gas-enriched areas, e.g. the Goliat oil-field within Ingøydjupet. The latter suggestion seems to be consistent with the fact that the studied core originates from the area with pockmarks. The values of Methylphenanthrene Index (MPI 1), an indicator of thermal maturity of OM, reach substantial values (up to 0.8), characteristic for hydrocarbons at the stage of mesocatagenesis (MC 1 -MC 2 ). The values of this parameter decrease down the core, which is probably related to the ratio between ancient and Holocene-Quaternary deposits in the sediments. However, there are three points of sharp deviation from the general trend, at the same depths as where the anomalies in hydrocarbon composition have been noted (21 cm, 136 cm, 223 cm), as described above. The significant decrease of MPI 1 observed in these samples points towards a considerable change in OM/hydrocarbon composition, namely to an input of significant amounts of weakly transformed, presumably biogenic, matter. As follows from the plot shown in Fig. 12, this process is most apparent in the upper part of the core (21 cm). It is also here that the odd-to-even predominance index of low molecular n-alkanes (OEP C 17-19 ) decreases sharply while the relative amounts of Te (22,29,30-trisnorhop-17(21)ene) increase (Fig. 12), which strongly supports the above-mentioned assumption.

Figure 12. A comparative characterisation of n-alkanes, triterpanes and MPI 1 (PAH) in the core from station 06JM-12.

Conclusions

A composite geochemical analysis of Holocene-Quaternary sediments from the area of pockmarks and their comparison with background sediments of South-Western Barents Sea have shown clear differences in the composition and distribution of hydrocarbon molecular markers in these sediments (Fig. 13).

It is notable that the sediments collected outside of the pockmarks field, both from the shelf and from deepwater (continental slope and its foot) areas, exhibit similar and uniform values of geochemical parameters indicating a common genesis and source of the original sedimentary OM. The slight differences observed between these samples are in accordance with existing knowledge on diagenetic transformation of bottom sediment OM in continental shelf and deeper sediments [START_REF] Degens | Factors controlling the distribution and early diagenesis of organic material in marine sediments[END_REF][START_REF] Yunker | Terrestrial and marine biomarkers in a seasonally ice-covered Arctic estuary -integration of multivariate and biomarker approaches[END_REF][START_REF] Fahl | Biomarkers as organic-carbon-source and environmental indicators in the Late Quaternary Arctic Ocean: problems and perspectives[END_REF][START_REF] Yamamoto | Late Pleistocene changes in terrestrial biomarkers in sediments from the central Arctic Ocean[END_REF][START_REF] Petrova | Geochemistry of organic matter of bottom sediments in the rises of the central Arctic Ocean[END_REF]2010). differences in the genesis and the level of transformation of OM. These differences hold for the whole sediment core, although they are manifested strongest at certain specific intervals, denoted as "anomalies" (Table 2). Thus, pockmark area sediments have a higher relative content and a higher level of transformation of low molecular weight n-alkanes (С 17 /С 27 ;

ОЕР С 17-19 ) at these horizons. A number of indicators point towards a higher level of thermal maturity of OM in these samples (22S/22S+22R C 31 ; МРI 1). At the same time they have higher contents of biogenic terpenoids, e.g. hopenes.

Table 2. Various organic geochemical parameters indicating the origin and maturity of organic matter in the studied sediments.

The observed combination of parameters may be explained by migration of the hydrocarbon fluid from deep strata, occurring in a pulsed manner and creating conditions favourable for intensive development of specific microbiota in situ. As the nutrient substrate is exhausted, and under the influence of diagenetic processes, the microbial activity is reduced, which is manifested in the levelling off of the geochemical parameters down the core. • We study geochemical biomarkers, as hopanes, in sediments from the Barents Sea.

• An area with pockmarks has a special hydrocarbon composition of sediments.

• The genesis of organic matter there seems to be petrogenic.

• The level of transformation and of thermal maturity of organic matter is high.

• This may be explained by migration of the hydrocarbon fluid from deep strata.

  Norwegian continental shelf seabed, has recently provided detailed geological and geochemical data on the composition of seabed sediments in South-Western

  fractions were analysed by GC-MS on a quadrupole Hewlett Packard 6850/5973 system. The analytical method described below has been certified in 2001 and 2005 by Russian State Standardization Service (Gosstandart), № РОСС RU.0001. 512785 of 18.07.2001.

  gradient from 50º С to 320º С at 3º С/min and 7 min at 320º С; full scan detection at 50 to 500 m/z, 70 eV. Saturated hydrocarbons (n-alkanes, isoprenoids) were analysed in selected ion monitoring mode (SIM) by ion mass m/z 71 (cyclic hydrocarbons), m/z 191 (hopanes) and m/z 217, 218 (steranes).Polycyclic aromatic hydrocarbons (PAH) were analysed on the following system: GC capillary column HP DB 30m x 0.25 mm x 0.25 µm, stationary phase 5%phenyl-95%methylpolysiloxane. Carrier gas helium, flow rate 1.2 ml/min; injector temperature 290ºС; oven temperature gradient from 60º С to 200º С at 20º С/min, to 300º at 10º С/min and 5 min at 300º С; full scan detection at 50 to 500 m/z, 70 eV.Parent PAH were analysed in selected ion monitoring mode (SIM) by ion masses m/z 128,

Figure 3 .

 3 Figure 3. The distribution of organic carbon (TOC) and total sulphur (total S) (a), the sum of aliphatic hydrocarbons (b), and the distribution of low molecular weight (C 14 -C 19 ) and high molecular weight (C 27 -C 31 ) n-alkanes normalised to TOC (c) versus depth in the gravity core from station 06JM-12.

Figure 5 .

 5 Figure 5. Ratios of low molecular weight n-alkanes (a), high molecular weight n-alkanes (b)and isoprenoids (c) in the gravity core from station 06JM-012.

  sample from 21 cm depth. This sample exhibits a practically monomodal distribution of n-alkanes with an clear maximum at С 16 . The predomination of low molecular weight hydrocarbons with a low OEP index (С 14 -C 19 /С 20 -C 35 = 0.77, ОЕР 17 = 0.7) is a highly specific signal of the presence of petroleum.

Figure 6 .

 6 Figure 6. The distribution of n-alkanes in the sediments from the continental shelf (a) and the pockmark area (b). Numbers for colored lines to the right in Figures 6a and -b are the sampled depths in cm.

Figure 9 .

 9 Figure 9. Hopanograms of the sediments from the core from the pockmark area (a) and from the background samples (b).

Figure 11 .

 11 Figure 11. The distribution of PAH in the core from station 06JM-12. Ph: Phenanthrene; Per: Perylene; Fl: Fluoranthene; MPI 1: Methylphenanthrene Index.

  At the same time, comparison of various geochemical parameters of the shelf sediments from within a pockmark area and from without (background samples) clearly indicates the

Figure 13 .

 13 Figure 13. A star-plot diagram showing the distinction between pockmark and background sediments.

Figure 1 .

 1 Figure1. Chemical structures of 22,29,30-trisnor-17α-hopane (Tm), 22,29,30-trisnor-18αhopane (Ts), and22,29,30-trisnorhop-17(21)-ene (Te).

Figure 2 .

 2 Figure 2. The positions of the sampling stations. Pink fields indicate pockmark locations while coloured fields indicate Goliat oil field.

Figure 3 .

 3 Figure 3. The distribution of organic carbon (TOC) and /total sulphur (total S) (a), the sum of aliphatic hydrocarbons (b), and the distribution of low molecular weight (C 14 -C 19 ) and high molecular weight (C 27 -C 31 ) n-alkanes normalised to TOC (c) versus depth in the gravity core from station 06JM-12.

Figure 4 .

 4 Figure 4. N-alkane distribution in the gravity core from station 06JM-012. Numbers for colored lines to the right are the sampled depths in cm.

Figure 5 .

 5 Figure 5. Ratios of low molecular weight n-alkanes (a), high molecular weight n-alkanes (b)and isoprenoids (c) in the gravity core from station 06JM-012.

Figure 6 .

 6 Figure 6. The distribution of n-alkanes in the sediments from the continental shelf (a) and the pockmark area (b). Numbers for colored lines to the right in Figures 6a and -b are the sampled depths in cm.

Figure 7 .

 7 Figure 7. N-alkane distribution in the sediments from the continental slope and its foot. Numbers for colored lines to the right are the sampled depths in cm.

Figure 8 .

 8 Figure 8. Mass-fragmentograms of n-alkylcyclohexanes distributions in various samples.

Figure 9 .

 9 Figure 9. Hopanograms of the sediments from the core from the pockmark area (a) and from the background samples (b).

Figure 10 .

 10 Figure 10. Triterpane distribution in the sediment core from station 06JM-12.

Figure 11 .

 11 Figure 11. The distribution of PAH in the core from station 06JM-12. Ph: Phenanthrene; Per: Perylene; Fl: Fluoranthene; MPI 1: Methylphenanthrene Index.

Figure 12 .

 12 Figure 12. A comparative characterisation of n-alkanes, triterpanes and MPI 1 (PAH) in the core from station 06JM-12.

Figure 13 .

 13 Figure 13. A star-plot diagram showing the distinction between pockmark and background sediments.

Table 1

 1

	, and the location of the sampling stations is shown in Fig. 2.
	The shorter sediment cores were collected in 2009 by NGU and IMR during MAREANO
	cruises in other areas of southwest Barents Sea and northeast Norwegian Sea, from
	continental shelf (342 water depth) to the bottom of the continental slope (2220 m) (see Table

Table 1

 1

	. A description of the analysed sediment samples from 4 locations.
	Figure 2. The positions of the sampling stations. Pink fields indicate pockmark locations
	while coloured fields indicate Goliat oil field.
	2.2. TOC, sulphur and grain size determination.

Table 1 .

 1 A description of the analysed sediment samples from 4 locations.

	Station	Date	Latitude,	Longitude,	Water	Lithology	Core	Sample dry
			N	E	depth, m		depth, cm	weight, g
	06JM-012	August 2006	71 o 19.38' 22 o 29.35'	432	silty clay	8-11
						silty clay	15-17
						silty clay	20-22
						silty clay	45-47
						silty clay	56.5-58.5
						silty clay	63-65
						silty clay	71-73
						silty clay	80-82
						silty clay	96-98
						silty clay	102-104
						silty clay	110-112
						silty clay	135-137
						silty clay	183-185
						silty clay	202-204
						silty clay	222-224
						silty clay	260-262
						clayey silt	270-272
						clayey silt	328-330
						clayey silt	419-421
	R406 MC032	April 2009 72 o 11.38' 14 o 49.61'	1035	Sandy silt	19-20
						Sandy silt	24-25
						Sandy silt	29-30
							32-33
	R422 BX090	April 2009 72 o 02.11' 16 o 54.74'	342	Silty sand	19-20
						No data	24-25
						No data	29-30
						No data	34-35
						No data	41-42
	R488 MC044	October 2009	69 o 42.89' 15 o 11.94'	2220	Sandy silt	14-15
						Sandy silt	19-20
						Sandy silt	24-25
						Sandy silt	29-30

Table 2 .

 2 Various organic geochemical parameters indicating the origin and maturity of organic matter in the studied sediments.

	Location	Shelf station R422BX90 mean Pockmark	Pockmark	Parameter
			mean	anomaly
	Ratios
	С 14-19 /С 20-35	0.39	0.35	0.77	Genesis indicator
	С 17 /С 27	1.68	1.34	7.49	Genesis indicator
	Pr/Ph	0.98	1.66	1.59	Facial /Genesis indicator
	ОЕР C 17-19	5. 04	1.04	0.74	Thermal maturity indicator
	Ts/Tm	0.45	0.17	nd	Thermal maturity indicator
	22S/22S+22R C 31	0.31	0.65	0.59	Thermal maturity indicator
	aβ/(aβ+βa) C 30	0.75	0.79	0.73	Thermal maturity indicator
	Te/Ts+Tm	0.69	2.03	7.36	Relative biogenic indicator
	hopene/Σhop	0.34	0.24	0.46	Thermal maturity indicator
	MPI 1	0.39	0.71	0.55	Thermal maturity indicator

Acknowledgements. We are grateful to the University of Tromsø for the sample material, to the Norwegian Ministry of Foreign Affairs for supporting the Norwegian-Russian cooperation, to Kjell Bakkeplass (IMR) for creating the map and to Penny Lee Liebig (IMR) for correcting the English.