Vidhyadevy Tangaraj

Jean-Marc Janot

Maguy Jaber
email: maguy.jaber@upmc.fr

Mikhael Bechelany

Sebastien Balme
email: sebastien.balme@umontpellier.fr

Adsorption and photophysical properties of fluorescent dyes over montmorillonite and saponite modified by surfactant

Keywords: montmorillonite, saponite, fluorescent dyes, adsorption isotherm, anisotropy

In the present study, the adsorption capacities of two intercalated smectites, CTAB-saponite and CTAB-montmorillonite with a cationic surfactant, were investigated with three fluorescent dyes namely Rhodamine 640 perchlorate rhodamine (Rho), sulforhodamine B (SR) and Kiton red 620 (KR). The adsorption isotherms fit well with the non-linear Langmuir isotherm model and the maximum adsorption capacities of all the composites are determined. The photophysical properties such as anisotropy and fluorescence lifetime of all the fluorescent dyes over the clay materials are determined. The set of experimental data based on X-Ray diffraction (XRD), transmission electron microscopy (TEM), Thermal analysis (TG-DTA) and fluorescence measurements allow highlighting the presence or the absence of interactions between the dyes and the modified clay minerals.

Introduction

The adsorption of dyes have been Longley investigated on clay minerals ( [START_REF] Thangaraj | Fluorescence Quenching of Sulforhodamine Dye over Graphene Oxide and Boron Nitride Nanosheets[END_REF][START_REF] Fournier | Physico-chemical characterization of lake pigments based on montmorillonite and carminic acid[END_REF]. The later have layered structure composed of octahedral and tetrahedral sheets consisting of fine particles of size ˂ 2 μm. Each tetrahedron consists of a cation, T, coordinated by four oxygen atoms and linked to adjacent tetrahedral by sharing three corners (the basal oxygen atoms, Oxb) to form an infinite two-dimensional 'hexagonal'. Each octahedron consists of a cation, O, coordinated by six oxygen atoms and linked to neighboring octahedral by sharing edges. They have been employed in several applications such as adsorbents, catalysts and host materials [START_REF] Jaber | Selectivities in Adsorption and Peptidic Condensation in the (Arginine and Glutamic Acid)/Montmorillonite Clay System[END_REF]. The surface properties of clay minerals were determined by the number of their active sites and the type of their exchangeable inorganic cations present in the interlayer space, their composition, the substitution ratio and its localization in the layers. These properties with the ability to swell in water and presence of both Bronsted and Lewis acidity make the clays possible excellent host materials for a wide range of organic and bio-molecules [START_REF] Raha | Photo-stability of rhodamine-B/montmorillonite nanopigments in polypropylene matrix[END_REF][START_REF] Tanabe | Solid acid and base catalysis In[END_REF][START_REF] Balme | Structure, orientation and stability of lysozyme confined in layered materials[END_REF][START_REF] Jaber | Mercaptopropyl Al-Mg phyllosilicate: Synthesis and characterization by XRD, IR, and NMR[END_REF][START_REF] Lepoitevin | BSA and lysozyme adsorption on homoionic montmorillonite: Influence of the interlayer cation[END_REF]. The Bronsted acidity is due to the formation of H + ions on the surface by dissociation of hydrated exchangeable metal cations of clays in water. This may also arise from a negative net charge on the surface by the substation of Al 3+ instead of Si 4+ at some tetrahedral positions and the net charge is balanced by H 3 O + cations.

Clays are acting as an electron acceptor in most of the electron transfer process and this Lewis acidity character arises due to the external surface of the clay build-up of outer surface and lateral surface (edges). In these cases mostly Al 3+ arising from rupture of Si-O-Al bond or Al 3+ at the edges, or through dehydroxylation of some Bronsted acid sites [START_REF] Wang | On different photodecomposition behaviors of rhodamine B on laponite and montmorillonite clay under visible light irradiation[END_REF][START_REF] Pankasem | Reflectance spectroscopic studies of the cation radical and the triplet of pyrene on alumina[END_REF]. The edges and the faces of clay particles are responsible to adsorb cations, anions, nonionic and polar contaminants from natural water. Adsorption of pollutants over the surface of clay may occur through ion exchange, ion-dipole interaction or coordination process from either strong or weak interactions [START_REF] Srinivasan | Advances in Application of Natural Clay and Its Composites in Removal of Biological, Organic, and Inorganic Contaminants from Drinking Water[END_REF][START_REF] El Adraa | Cysteine-montmorillonite composites for heavy metal cation complexation: A combined experimental and theoretical study[END_REF].

Industries are main source of water pollution and it produces toxic pollutants that are harmful to humans, animals and water bodies. One of such important pollutant is dyes; colored compounds are widely used in many industries such as textile, leather, pharmaceutical, paper, tanning, pulp etc. Some of dyes are carcinogenic and not biodegradable [START_REF] Vidhyadevi | Adsorption of Congo Red Dye over Pendent Chlorobenzylidine Rings Present on Polythioamide Resin: Kinetic and Equilibrium Studies[END_REF][START_REF] Premkumar | Kinetic and equilibrium studies on the biosorption of textile dyes onto Plantago ovata seeds[END_REF]. Various physicochemical and biological treatment methods have been reported to remove organic dyes from wastewater.

Adsorption is proven to be a practical way for the treatment of dye wastewater due to its efficiency, selectivity and cost effective nature [START_REF] Vidhyadevi | Optimization of the process parameters for the removal of reactive yellow dye by the low cost Setaria verticillata carbon using response surface methodology: Thermodynamic, kinetic, and equilibrium studies[END_REF]. The modification of clay minerals with organic molecules such as surfactant [START_REF] He | Organoclays prepared from montmorillonites with different cation exchange capacity and surfactant configuration[END_REF][START_REF] Guegan | Intercalation of a Nonionic Surfactant (C10E3) Bilayer into a Na-Montmorillonite Clay[END_REF] has been widely reported to increase the affinity of these materials with organic pollutants [START_REF] Nourmoradi | Removal of benzene, toluene, ethylbenzene and xylene (BTEX) from aqueous solutions by montmorillonite modified with nonionic surfactant: Equilibrium, kinetic and thermodynamic study[END_REF][START_REF] Sanchez-Martin | Efficiency of different clay minerals modified with a cationic surfactant in the adsorption of pesticides: Influence of clay type and pesticide hydrophobicity[END_REF][START_REF] Klumpp | Synergistic Effects between Cationic Surfactants and Organic Pollutants on Clay-Minerals[END_REF].

Among different types of dyes, molecules of Xanthene family are commonly used for their photophysical properties and have vast applications in sensors based thin films, in biological systems, in laser technology, in supramolecular chemistry etc. The three fluorescent dyes (Figure 1) namely rhodamine 640 (Rho), sulforhodamine 640 (SR) and Kiton red 620 (KR) are selected for the current study due to their very good quantum efficiency and their photochemical stability.

Preparation of clays

Cation-exchange Montmorillonite and saponite:

Na-Mt or Na-Sap was exchanged with cetyltrimethylammonium bromide (CTAB). The clay mineral was added in a large amount of the surfactant solution (0.1 mol L -1 ) and kept under stirring. A ratio of 1.5 g of montmorillonite for 100 mL of solution was chosen. Three successive baths of increasing times were conducted under stirring, 1 hours, 3 hours, and 24 hours to perform a total cationic exchange. After the final bath of 24h, samples were recovered by centrifugation and five washing steps with distilled water. After filtration, the solid fraction was dried in an oven at 60°C. The obtained materials are noted CTAB-Mt and CTAB-Sap.

Dye adsorption on material

The fluorescence dyes were adsorbed on the materials as follow: 2 mg of clays was added to 2 mL of dye solution at the different concentration (from 0.25 to 8 mg L -1 ). The mixture was stirred for one hour at room temperature and then centrifuged to separate the adsorbent. The concentration of dye in supernatant was analyzed using UV-visible spectrophotometer (JASCO).

The dye concentration was obtained from the absorbance at 566 nm for Kiton Red 620, 574 nm for rhodamine 640 and 586 nm for sulforhodamine 640.

Instrumental methods

All the samples were characterized by Fourier Thermogravimetric Analysis (TGA) using TA instruments TGA G500 under oxygen flow of 60 mL min -1 . The samples were heated up to 700 °C at a heating rate of 10 °C min -1 and equilibrated for 60 minutes at 700 °C for complete degradation of organic molecules. X-ray diffraction (PANAlyticalXpert-PRO diffractometer equipped with aX'celerator detector using Ni-filtered Cu-radiation) were recorded using Cu-K α (λ =1.5418 Å) radiation at 40 kV and 20 mA and secondary monochromator in the 2Ɵ range of 3-70° with a scan speed of 2° min -1 . Transmission electron microscopy (TEM) study of the samples was performed on a JEOL100CX microscope. Samples in the form of bulk powders were suspended in ethanol and then deposited on 400 mesh copper grids covered with an ultrathin carbon membrane of 2-3 nm thickness.

Time resolved fluorescence analysis

For Fluorescence experiments, 2 mL of the 10 mg L -1 of each fluorescent dye solution was mixed with 2 mg of clays. The mixture was stirred and analyzed after one hour using fluorimeters. The steady-state fluorescence were recorded by fluorescence spectrophotometer Spex (Fluorolog 1681) at a scanning rate of 1 nm s -1 . Time-resolved fluorescence data were obtained by the time-correlated single-photon counting technique [START_REF] Balme | Highly efficient fluorescent label unquenched by protein interaction to probe the avidin rotational motion[END_REF][START_REF] O'connor | Time Correlated Single Photon Counting[END_REF]. The excitation wavelength was achieved by using a SuperK Extrem high power white supercontinum laser (NTK Photonics, model EXR-15) as a continuum pulsed source. The wavelength was selected by coupling to a monochromator (Jobin-Yvon H10). The repetition rate was set to 38.9 MHz; the excitation pulse duration on this device is around 6 ps (full-width-at-half-maximum, FWHM).

The emission of fluorescence is detected, after passing through a polarizer oriented parallel, perpendicular or at the magic angle (54.73°) to polarization of excitation, through a double monochromator Jobin-Yvon DH10 on a hybrid PMT detector HPM-100-40 (Becker &Hickl).

The instrumental response function of the equipment was measured by using a dilute suspension of polystyrene nanospheres in water (70 nm of diameter) as a scattering solution; it was typically about 130-160 ps FWHM. Decays were collected at a maximum count rate of 15 kHz into 4096 channels using an acquisition card SPC-730(Becker & Hickl). This limiting count rate was achieved by dilution of the sample and after sedimentation of the suspension in order to minimize as much as possible the scattering of the particles. The time per channel was set around 6 ps ch -1 in order to fit a full decay in the experimental time window. All decays were collected so as to have at least 1.5 10 6 counts in total. Decay analysis was performed using a Levenberg-Marquardt algorithm. For the analysis, the fluorescence decay law at the magic angle I M (t) were assumed as a sum of exponentials. Fluorescence lifetimes were calculated from data collected at magic angle by iterative adjustment after convolution of a pump profile (scattered light) with a sum of exponentials. We assumed a Poisson distribution of counts in the calculation of the χ 2 criterion; residuals profiles and autocorrelation function as well as Durbin-Watson and skewness factor were used in order to estimate the quality of the adjustment. The number of exponentials used for the fit was increased until all the statistical criterions were improved.

Results and discussion

TGA Analysis

The thermal stability of all the samples is shown in Figure 2a The DTG curves (Figure 2b) of CTAB-Mt and CTAB-Sap samples show the peaks of montmorillonite and saponite plus some specific signals with maxima at about 240 and 400 °C, which are assigned to the progressive elimination of the organic matter. The first degradation is attributed to the loss of water and solvent from the ambient to 190-200˚C temperature range. The second step is from 190˚C to 275˚C and is due to the loss of hydration water from the Na + ion. In the third degradation step, at the temperature at around 410˚C is assumed to be the surfactant removal. The loss structure of the hydroxyl group within the clay is occurred at the final degradation step in the TGA curves at around 540˚C. This result confirms the thermal stability of modified montmorillonite and saponite clays by surfactant previously reported elsewhere [START_REF] Beall | Self-assembly of organic molecules on montmorillonite[END_REF][START_REF] Xi | Modification of Wyoming Montmorillonite Surfaces Using a Cationic Surfactant[END_REF].

XRD analysis

XRD patterns (Figure 3) of synthetic montmorillonite and saponite exhibit typical hkl bands of smectites [START_REF] El Adraa | Adsorption of L-DOPA Intercalated in Hydrated Na-Saponite Clay: A Combined Experimental and Theoretical Study[END_REF][START_REF] Lambert | A comparative study of the catalysis of peptide bond formation by oxide surfaces[END_REF]. The (001) reflexion shows an inter-reticular distance of 1.26 nm, typical of the "one-water layer" form. The d (060) of the Mt is equal to 0.149 nm typical of dioctahedral character while the one of saponite is equal to 0.152 nm characteristic of trioctahedral character.

TEM experiments

Transmission electron microscopy (TEM) micrographs show layered structures with alternate dark and bright fringes with a repeat length of 1.2 nm for the raw Mt. After Sr adsorption, an heterogeneity in the stacking of the layers is observed confirming the absence of the (001) reflexion in the XRD pattern. In the CTAB-Mt samples, a distance of 2.2 nm (Figure 4) is measured. For and SR-CTAB-Sap samples, TEM micrographs exhibit an heterogeneity in the layer stacking explaining the absence of (001) reflexion in the XRD patterns: indeed images show some exfoliated layers and others intercalated with organic molecules with a d(001) spacing of 2.5 nm. (Figure 4).

Isotherm studies

Adsorption isotherm models play an important role in the determination of the maximum capacity of adsorption, and clearly depict the relationship of the amount of dyes adsorbed by a unit weight of the clays at equilibrium. The equilibrium adsorption isotherm explains how the dye interacts with the clay, and how the adsorbate distributes between the solution (C e ) and the solid phase (q e ) when the adsorption process reaches the equilibrium state. Adsorption isotherm models such as Langmuir [START_REF] Langmuir | The Adsorption of Gases on Plane Surfaces of Glass, Mica and Platinum[END_REF] and Freundlich [START_REF] Freundlich | Over the adsorption in solution[END_REF] were commonly used to describe the experimental data [START_REF] Benetoli | Adsorption of nucleic acid bases on clays: an investigation using Langmuir and Freundlich isotherms and FT-IR spectroscopy[END_REF].

The Langmuir adsorption isotherm is based on the assumption that the monolayer adsorption onto a surface containing a finite number of adsorption sites. It also assumes uniform energies of adsorption as well as no mutual interaction between the adsorbed molecules. The non-linear equation of the Langmuir isotherm model is expressed by equation 1:

(1)

𝐪 𝐞 = 𝐪 𝐦 𝐤 𝐋 𝐂 𝐞 𝟏 + 𝐤 𝐋 𝐂 𝐞
where q m is the maximum monolayer adsorption capacity, K L the Langmuir equilibrium constant.

The essential characteristics of the Langmuir isotherm parameters can be expressed in terms of a dimensionless constant called the separation factor, or a dimensionless equilibrium parameter, "R L " [START_REF] Vidhyadevi | A study on the removal of heavy metals and anionic dyes from aqueous solution by amorphous polyamide resin containing chlorobenzalimine and thioamide as chelating groups[END_REF]. The dimensionless constant R L is the essential characteristic of the Langmuir isotherm and it can be expressed by equation 2(2)

𝐑 𝐋 = 𝟏 𝟏 + 𝐤 𝐋 𝐂 𝐨
The Freundlich isotherm is based on the assumption that adsorption occurs on a heterogeneous adsorption surface with different energies of adsorption and is given by the equation 3.

(3) 𝐪 𝐞 = 𝐊 𝐟 . 𝐂 𝟏/𝐧 𝐞 where K f and n are the Freundlich constants related to the adsorption capacity and adsorption intensity respectively. The value of 1/n that varies between 0.1 to 1.0 indicates the favorable adsorption of molecule. n is a measure of the deviation of the adsorption isotherm linearity. The experimental data (q e and C e ) were fitted to above isotherm models using MATLAB 7.1 on the effect of an initial concentration of Rho, SR and KR ions on the clays, and the graphical representations of these models are presented in Figures 5. The results are reported on Table 1 and 2 for Langmuir and Freundlich model respectively. The dye adsorption on clay with or without surfactant can be roughly described by Langmuir isotherm following the value of R 2 . It seems that the presence of CTAB has not clear influence on q m . However it decreases the Langmuir constant affinity for Rho and KR. For SR, the results suggest a better affinity with CTAB exchanged samples in the case of Sap. The calculated R L values based on the Langmuir adsorption isotherm are close to 1 in a adsorption capacity q m range of 8 to 20 mg g -1 , indicating that the adsorption process is not strongly favorable. The values of adsorption capacity are lower than the one reported for rhodamine 6G 38.21 mg g -1 [START_REF] Selvam | Removal of rhodamine B from aqueous solution by adsorption onto sodium montmorillonite[END_REF]. This should be explained by the ability to the rhodamine G for form dimer [START_REF] Klika | The rhodamine B intercalation of montmorillonite[END_REF].

CLAYS

Freundlich parameter n value indicates the degree of non-linearity between the solution concentration and adsorption as follows: When n = 1, adsorption is linear, adsorption sites are homogeneous in nature, no further adsorption takes place at the same site; when n < 1, adsorbates are bound with weaker energies with adsorption sites. On the contrary, when n > 1, more adsorbates are present in the solution enhances free energies for further adsorption. In the present study, the value of n was greater than 1 for all samples, which indicates the non-linear heterogeneous adsorption of KR, SR, Rho.

CLAYS

Impact of clays on photophysical properties of fluorescent dyes

The photophysical properties of the different clay samples were studied by time resolved fluorescence in aqueous solution. The fluorescence decay was reported in Figure 6 and the results are summarized in Table 3. For KR, SR and Rho in water solution, the decay is fitted by monoexponential function with a lifetime τ KR =1.58 ns, τ SR = 4 ns and τ RHO = 4.1 ns respectively.

These three dyes have a close chemical structure which explains the similar lifetime for SR and Rho. The shorter lifetime for KR is usually explained by the dimer structure. When dyes are adsorbed on the different clays the fluorescence decay fit required two or three exponential components. For Rho, the long lifetime around 4 ns represents more than 90 % yield whatever the clay. This suggests that the dye is surrounded by water molecules and thus not adsorbed on the clay surface. This is confirmed by the correlation time which is the same than the one for Rho free in solution. The second lifetime is shorter from 0.85 to 1.5 ns and depends on the clay sample. Indeed it is longer for sample exchange with CTAB than the one exchange with Na + or Mg + . This second lifetime could be assigned to the low amount of dye adsorbed on the material.

The SR follows a similar behavior that Rho: (i) two lifetimes are required to fit the fluorescence decay, (ii) the long lifetime is the one than the dye in solution and (iii) the short lifetime is longer for clay exchange with CTAB. However in the latter case, the yields of the short lifetime are 18% and 36.4% while less than 5 % for the Rho. This could be explained by the better affinity of SR for CTAB modified clay than Rho which is in good agreement with the adsorption isotherm results (Table 1-2). The fluorescence decay of adsorbed KR require also 2 lifetimes one around 3.5 ns and one around 1.5 ns. As the other sample, the correlation reveals that most of dye molecules are free in water solution. As previously mentioned, the KR is in dimer form which explains the short lifetime around 1.5 ns. The lifetime around 3.5 ns could be assigned to KR monomer in water solution. This means that after adsorption, the clay permits a release of KR monomer. The separation of monomer is more efficient for Na-Sap and CTAB exchanged clay than for Na-Mt. Compare to the SR and Rho, the correlation with the affinity and the yield of short lifetime cannot be done. Indeed, the populations as dimer in solution than the one adsorbed on clay cannot be separated. Globally the fluorescence lifetime experiments permit to show the release of fluorescence dye. This is in good agreement with the low affinity between the clay and the dye as well as the location of dye at the surface of grain.

Conclusion

Two different clays Na-Mt and Na-Sap were prepared and exchange with cationic surfactant CTAB. Thermogravimetric analysis confirmed the presence of the organic matter in both clay minerals. The adsorption isotherm results showed that the affinity of dye is limited for interfacial concentration range from 7 mg g -1 to 20 mg g -1 . The XRD patterns indicate an intercalation of the CTAB in the interlayer space. The presence of dyes does not affect the d001 value indicating that no intercalation occurred in interlayer space due probably to the low concentration of the dye in the material. The time resolved fluorescence of the adsorbed dyes show that most of the dye molecules are released in solution. This is due likely to the poor affinity with the clay minerals and the location at the grain surface. Interestingly in the case of KR which is usually in dimer form in water, the adsorption on clay permits to release monomer in solution characterized by a fluorescence lifetime around 3 ns. Regarding these results, we can conclude that the clay could be interesting to dissociate this dye before using in water solution.

Acknowledgement

  The aim of the present work is to study the efficiency of different smectites in the adsorption of three fluorescent dyes i.e. Rho, SR, and KR. The smectites used were synthetic sodium montmorillonite (Na-Mt) and sodium Saponite (Na-Sap) and their equivalent modified with cetyltrimethylammonium bromide, a cationic surfactant (CTAB). The dyes location has been studied by XRD and TEM. The adsorption isotherms have been conducted to evaluate the adsorption mechanism and to evaluate the adsorption capacities of the clays. The photophysical properties of the adsorbed dyes were investigated in aqueous solution by the time resolved fluorescence and anisotropy of fluorescence.

Figure 1 :

 1 Figure 1 : Structure of dyes used in this study

  . Results show that unmodified clays such as Na-Mt and Na-Sap undergo three stages of degradation. The first stage involves a weight loss of 6 -9 % in the temperature range of 45-70ºC, which is due to the evaporation of the physisorbed water. The second stage of degradation is assumed to be dehydration of the hydrated cation in the interlayer at the temperature range of 80-150ºC with a weight loss of 15 %. A weight loss of 3 % for Na-Mt and 0.2 % for Na-Sap occurs in the third stage, in the temperature range of 450-650ºC, which is due to the dehydroxylation of the corresponding clays.

Figure 2 :

 2 Figure 2 : a) TGA curves of raw Na-Mt, Na-Sap, CTAB-Mt and CTAB-Sap and b) DTG curves of raw Na-Mt, Na-Sap, CTAB-Mt and CTAB-Sap.

  The direct adsorption of the dye on the Na-Sap clay mineral does not induce any change. On the opposite, for Na-Mt, the d(001) disappeared after SR adsorption. This should be explained by a disordered stacking of the Mt layers. After exchange with the cationic surfactant, the d 001 value increases until 2.26 nm and 2.5 nm, indicating the intercalation of the later in the interlayer space of Mt and Sap, respectively. The adsorption of the anionic dye induce the disappearance of the peak at 3.5°(2) in the case of CTAB-Mt which can correspond to the desorption of the cationic surfactant by the dyes. In the case of CTAB-Sap sample, does not show any modification on the XRD pattern for both CTAB-Mt and CTAB-Sap. The XRD of the SR-CTAB-Sap does not exhibit any peaks in the region 2-10° (2) due probably to a partial exfoliation or a heterogeneity in the layers stacking.

Figure 3 :

 3 Figure 3 : XRD patterns of a) Na-Sap, Na-Sap, CTAB-Sap, SR-CTAB-Sap and b) Na-Mt, SR-Mt, CTAB-Mt, SR-CTAB-Mt

Figure 4 :

 4 Figure 4 : TEM micrograph of raw Na-Mt (a), SR-Mt (b), SR-CTAB-Mt (c-d) and for SR-CTAB-Sap (e-f)

Figure 5 :

 5 Figure 5 : Adsorption isotherm of KR (a-b), SR (c-d) and Rho (e-f) onto Na-Sap, Na-Mt, CTAB-Sap and CTAB-Mt. The open symbols are the experimental points. The lines are the nonlinear fit using Langmuir (a-c-e) and Freundlich (b-d-f) model

Figure 6 : 1 (

 61 Figure 6: Examples of fluorescent decay Kr (red), SR (Blue) and Rho (black) loaded on Na-Mt CLAYS τ 1 (Yield) (ns) τ 2 (ns) (Yield) τ 3 (ns) (Yield) χ 2

Free

  introduced into a PTFE-lined stainless steel autoclave. The autoclaves were heated at 200°C for 72 h. After reaction, autoclaves were cooled to room temperature. Run products were then recovered by filtration, washed thoroughly with distilled water and dried at 60°C for 12 h.

	0.1•Na 2 O : 0.05•HF : 96•H 2 O[21]. The hydrogels were matured over a 2 h period at room
	temperature and then introduced into a PTFE-lined stainless steel autoclave. The autoclaves were
	heated at 200°C for 72 h. After reaction, autoclaves were cooled to room temperature. Run
	products were then recovered by filtration, washed thoroughly with distilled water and dried at
	60°C for 12 h. The ideal formula per half unit cell is Na 0.2 (Si 4 )(Al 1.8 Mg 0.2 )O 10 (OH,F) 2 ] with a
	theoretical octahedral substitution rate (i.e. the number of Mg 2+ to Al 3+ substitution per half-cell)
	of 0.2, yielding a cation exchange capacity of 0.382 meq g -1 [22].
	Saponite was synthesized according to a previously published procedure having the following
	chemical formula: Na 0.4 [Si 3.6 Al 0.4 )(Mg 3 )O 10 (OH, F) 2 ]. That is, the Al substitution rate expressed
	per full unit cell is x =0.8 [23]. Hydrofluoric acid (HF, 40% w/w; Fluka), sodium acetate
	(NaCOOCH 3 , 99%; Fluka), magnesium acetate (Mg(COOCH 3 ) 2 4H 2 O, 99%; Fluka), aluminum
	basic diacetate (19%, sigma aldrich), and silica (SiO 2 , 99.5%, Aerosil 130; Evonik) were used to
	prepare a hydrogel with the following chemical composition 1SiO 2 : 0.15•Al 2 O 3 : 0.2•MgO :
	0.1•Na 2 O : 0.05•HF : 96•H 2 O. The hydrogels were matured over a 2 h period at room
	temperature and then
	Montmorillonite synthesis: Hydrofluoric acid (HF, 40% w/w; Fluka), sodium acetate
	(NaCOOCH 3 , 99%; Fluka), magnesium acetate (Mg(COOCH 3 ) 2 4H 2 O, 99%; Fluka), boehmite
	(Al 2 O 3 , 75278%, Pural SB1; Condea), and silica (SiO 2 , 99.5%, Aerosil 130; Evonik) were used
	to prepare a hydrogel with the following chemical composition 1SiO 2 : 0.15•Al 2 O 3 : 0.2•MgO :

Table 1 : Langmuir Isotherm Parameters for the adsorption of KR, SR AND Rho over clays

 1

			Na-Mt	CTAB-Mt	Na-Sap	CTAB-Sap
	KR	K L (L mg -1 )	0.385	0.245	0.214	0.093
		q m (mg g -1 )	17.29	18.42	19.90	13.46
		R L	0.24-0.91	0.82	0.84-0.88	0.92
		R 2	0.804	0.925	0.988	0.818
	SR	K L (L mg -1 )	0.925	0.190	0.116	0.375
		q m (mg g -1 )	10.93	10.97	12.58	19.21
		R L	0.81-0.12	0.85	0.89-0.9	0.75
		R 2	0.893	0.984	0.981	0.978
	Rho	K L (L mg -1 )	0.977	0.128	0.071	0.010
		q m (mg g -1 )	7.93	8.78	16.78	12.22
		R L	0.8-0.11	0.89	0.93-0.95	0.99
		R 2	0.975	0.824	0.945	0.943

Table 2 : Freundlich Isotherm Parameters for the adsorption of KR, SR and Rho over clays

 2

			Na-Mt	CTAB-Mt	Na-Sap	CTAB-Sap
	KR	K f (mg g -1 )	1.851	3.881	3.381	1.841
		n	4.242	4.678	4.561	3.242
		R 2	0.8314	0.8655	0.9792	0.9485
	SR	K f ( mg g -1 )	3.739	3.726	4.166	4.879
		n	1.837	3.047	2.846	1.156
		R 2	0.8003	0.935	0.945	0.9825
	Rho	K f ( mg g -1 )	2.114	2.966	4.8825	3.389
		n	1.863	1.103	3.639	2.317
		R 2	0.991	0.896	0.965	0.951

Table 3 : Fluorescent lifetime (τi) and yield for dye loaded in Mt Al (Yield = 100αiτi/Σαiτi represents the contribution of each emission to the total emission)

 3

Dr. Vidhyadevi Thangaraj is thankful to the Erasmus Mundus-Svagata, European

Commission for providing a fellowship for this research.

Na-Mt and Na-Sap were prepared and exchange with cationic surfactant CTAB The adsorption of Rhodamine, kiton red and sulforhodamine on these materials was studied Whatever the material the fluorescent dyes are not located inside interlamellar space The dyes are released in water as monomer even for the kiton red


G Smolyakov

C Formosa-Dague

C Severac

R E Duval

E Dague

High speed indentation measures by FV, QI and QNM introduce a new understanding of bionanomechanical experiments

Keywords:

Structural and mechanical mapping at the nanoscale by novel high-speed multiparametric Quantitative Imaging (QI) and PeakForce Quantitative Nanomechanical Mapping (PF-QNM) AFM modes was compared to the classical Force Volume (FV) mapping for the case of living Pseudomonas aeruginosa bacterial cells. QI and PF-QNM modes give results consistent with FV for the whole cells in terms of morphology and elastic modulus, while providing higher resolution and shorter acquisition time. As an important complement, the influence of scanning parameters on elastic modulus values was explored for small 0.2 2 µm 2 central area on top of cells. The modulus decreases with the indentation depth due to the effect of the hard cell wall, while it increases versus tip oscillation frequency, displaying viscoelastic behaviour of the living bacterial cells. The ability of different AFM modes to follow correctly the bacteria viscoelastic behaviour at high oscillation frequency was tested.

Introduction

Atomic force microscopy [START_REF] Binnig | Tunneling through a controllable vacuum gap[END_REF][START_REF] Binnig | Atomic force microscope[END_REF] has become a powerful tool for structural and mechanical mapping of biological samples at the nanoscale [START_REF] Muller | Force probing surfaces of living cells to molecular resolution[END_REF]Dufrêne et al., 2008;[START_REF] Cross | Nanomechanical analysis of cells from cancer patients[END_REF][START_REF] Liu | Application of AFM in microbiology: a review[END_REF][START_REF] Dorobantu | Application of atomic force microscopy in bacterial research[END_REF]Formosa et al., 2012a;Formosa et al., 2012b;[START_REF] Yao | Atomic force microscopy and theoretical considerations of surface properties and turgor pressures of bacteria[END_REF][START_REF] Aguayo | Single-bacterium nanomechanics in biomedicine: unravelling the dynamics of bacterial cells[END_REF]. Since the development of the Force Volume (FV) mode in 1994 [START_REF] Radmacher | Mapping interaction forces with the atomic force microscope[END_REF], it became possible to obtain force curves related to the interaction between the AFM tip and the sample surface. These curves give access to the surface topography and mechanical properties simultaneously. In particular, sample deformation and elastic modulus can be extracted, as shown in Fig. 1a. For that, the approach force curve is fitted with an appropriate geometric model. On the other hand, the retract curve may be used in order to calculate the adhesion force. The latter corresponds to the minimum force and may have nonspecific or specific nature.

In the FV mode, the tip approaches the surface until a certain force, called peak force, is reached. Then the tip is retracted and moves to the next position (Fig. 1b). Recently a new solution has been proposed by Bruker Corporation: a high-speed multiparametric mode called PeakForce Quantitative Nanomechanical Mapping (PF-QNM) (Hu et al., 2011;[START_REF] Alsteens | High-Resolution Imaging of Chemical and Biological Sites on Living Cells Using Peak Force Tapping Atomic Force Microscopy[END_REF][START_REF] Dufrêne | Multiparametric imaging of biological systems by force-distance curve-based AFM[END_REF][START_REF] Martín-Rodríguez | On the influence of the culture conditions in bacterial antifouling bioassays and biofilm properties: Shewanella algae, a case study[END_REF]. In this mode the tip pathway is modified, allowing faster scans. The height and mechanical properties images are provided in real time, while the registration of force curves is an additional option. In its turn, JPK Instruments has also developed a multiparametric mode, called Quantitative Imaging (QI) (Chopinet et al., 2013a;Chopinet et al., 2013b). Analysis of the saved force curves in this mode gives access to the sample height and mechanical behaviour. A comparison of FV, PF-QNM and QI modes is given in Fig. 1c. The PF-QNM mode works at the highest oscillation frequency and can adopt a number of discrete values from 0.25 kHz to 8 kHz.

For biological research a relatively low frequency of 0.25 kHz or 0.5 kHz is usually applied. In the FV and QI modes the frequency range is about 0.5÷100 Hz. On the other hand, the minimum resolution in FV and QI is much lower (8 2 px 2 ) compared to PF-QNM (128 2 px 2 ), thus allowing very fast scans, if detailed surface map is not required. In its turn, PF-QNM is the only mode in which a good statistics for every pixel in terms of number of force curves is possible. Resulting value for a pixel represents an average from several force curves. This is very useful for nanomechanical or nonspecific adhesion investigations, while it has no sense for specific adhesion measurements, as the average value will depend on the probability of specific adhesion events. Thus in order to measure the specific adhesion value correctly, the PF-QNM parameters should be adapted in the way to have only one force curve per pixel. On the other hand, very high speed of PF-QNM may prevent a specific bond establishment, as the latter takes some time. As for the FV and QI, in the latter modes only one force curve contributes to each pixel.

Pseudomonas aeruginosa (Pa) is an invasive, Gram-negative pathogen that causes a wide range of severe infections including bacteraemia, pneumonia, meningitis, urinary tract and wound infections [START_REF] Hauser | Pseudomonas aeruginosa[END_REF]. PaR3 (kindly provided by Pr. R.E. Duval from ABC Platform, Nancy, France) has been taken as an object for the present study, being a clinical multiresistant strain (antibiogram is shown in Supplementary data 1).

The main aim of the present work was to apply Nanowizard III (JPK Instruments) and Bioscope Catalyst (Bruker Corporation) instruments for elucidating the nanomechanical behavior of living bacterial cells when indented at high frequency with AFM. The study was limited to these two instruments, although other manufactures propose as well the solutions for probing the bionanomechanical properties. We used the classical FV mode and the novel high-speed QI and PF-QNM modes. The work has been performed using the following strategy: first, the whole bacteria were explored by the three AFM modes in order to compare the cell morphology, as well as the elastic modulus variation across the cells; second, small 0.2 2 µm 2 zones in the centre of the cells were scanned in order to see a dependence of the elastic modulus from major scanning parameters: applied force and oscillation frequency; third, the possibility of very fast FV scans (classically considered as a slow mode) was explored with both JPK and Bruker instruments, using bacteria as well as standard PDMS samples.

Materials and methods

Bacterial sample preparation

PaR3 were stocked at -80 o C, revivified on Mueller Hinton agar (Difco, 225250-500 g) and grown in 5 mL of Mueller Hinton broth (Difco, 275730-500 g) for 24 h at 35 o C under static conditions. Then 10 µL of the cells suspension were introduced to 5 mL of Mueller Hinton broth for 24 h at 35 o C under static conditions.

The same procedure was repeated one more time for a good standardization of bacterial cell cultures. PaR3 cells were then immobilized on polyethylenimine (PEI, Fluka P3124-100 mL) coated glass slides as described previously [START_REF] El Kirat | Sample preparation procedures for biological atomic force microscopy[END_REF]. Briefly, freshly oxygen plasma activated glass slides were covered by a 0.2% PEI solution in deionized water and left for incubation overnight. Then glass slides were rinsed with 10 mL of Milli-Q water and nitrogen dried. Bacterial cultures were harvested by centrifugation, washed two times in PBS, and resuspended to a concentration of ~10 8 cells/mL in phosphate buffered-saline 1X (10 mM) (PBS, Sigma, P2194-10PAK). 1 mL of the suspension was deposited on the PEI coated glass slide and allowed to stand for 20 min before rinsing with PBS 1X.

Atomic force microscopy

Two instruments were used for the present study: Nanowizard III (JPK Instruments) and Bioscope Catalyst (Bruker Corporation). As for the scanning conditions for both bacterial and PDMS samples, ramp size was set to 500 nm, peak force to 1 nN, except for the experiment with increased penetration depth close to the cell diameter, for which the peak force was increased up to 8 nN. The oscillation frequency was set to 6 Hz for FV, 40 Hz for QI and 250 Hz for PF-QNM (if not specified otherwise). The images resolutions were 32 2 px 2 for FV and 128 2 px 2 for QI and PF-QNM modes. Measurements were done in a physiological PBS 1X buffer, using MLCT-C cantilever (if not specified otherwise) with a nominal spring constant of 10 mN/m (with an average value of 12.5 mN/m). The thermal tuning method [START_REF] Emerson Iv | On the importance of precise calibration techniques for an atomic force microscope[END_REF] was used to calibrate the spring constant of the cantilever before each experiment.

The SPIP software was used for the analysis of the FV and QI results, with the force curves correction stage consisted in the noise reduction and the base line adjust. As for the PF-QNM, this mode provided the results in real time during the experiment.

Elastic model

Hertz elastic model for spherical contact between tip and surface [START_REF] Hertz | Über die berührung fester elastischer Körper (On the contact of rigid elastic solids)[END_REF] was chosen for the analysis of the FV and QI data. MLCT (Bruker Corp.) cantilevers with a nominal tip radius of 20 nm were used for the present study. Cell maximum deformation at peak force was about 40 nm corresponding to an average deformation around 20 nm along the fitted part of the force curve. Although for the average deformation close to the tip nominal radius both Hertz and Sneddon [START_REF] Sneddon | The relation between load and penetration in the axisymmetric boussinesq problem for a punch of arbitrary profile[END_REF] models are valid, we chose the Hertz one for the best fit of the small indentation (lower than the tip nominal radius) part of the force curve. The Hertz model links the sample deformation d to the applied force F via elastic modulus E and tip radius R values:

F = 4/3*E/(1 -ν 2 )*R 1/2 *d 3/2 ,
with ν being sample Poisson ration, arbitrary taken as 0.5.

The use of the MLCT-C cantilevers and 1 nN as the force set point resulted in the maximum cantilever deflection around 80 nm, that is about two times larger compared to the sample deformation. This allowed precise subtraction of cantilever deflection from the whole piezoelectric movement. On the other hand, the cantilever was highly sensitive allowing following in details the shape of force curves.

Instead of performing the tip radius calibration on a standard sample, which can damage or contaminate the tip, the same cantilever was used for different modes to account for a possible error in the tip radius value.

As for the absolute modulus values, the latter have rather weak dependence from the tip radius (E ~ R -1/2 ), in addition these modulus values are relative by the AFM technique in principle.

As the PF-QNM provides the results in real time and the Hertz model is not embedded in the Bruker's online software, thus the Sneddon model was used for the modulus fit. The Sneddon model operates with the tip half-angle α (equal to 17.5 o for the MLCT-C cantilevers) as the geometric parameter of the tip:

F = 2/π*E/(1 -ν 2 )*tanα*d 2 .
As the sample deformation is increased in the PF-QNM compared to the QI or FV, the use of the Sneddon model for the PF-QNM measurements is indeed reasonable. The application of both Hertz and Sneddon models for the FV data showed that the difference between the modulus values by two models is as low as 10% (which shows again that both models could be applied). This difference in modulus by two models is much lower compared to the differences between AFM modes, which are discussed in this article.

So the bacterial cells mechanics is considered within the elastic approach in this work, without quantification of viscoelastic contribution, which may be important in real bacterial cells [START_REF] Vadillo-Rodriguez | Surface viscoelasticity of individual gram-negative bacterial cells measured using atomic force microscopy[END_REF][START_REF] Vadillo-Rodriguez | In situ characterization of differences in the viscoelastic response of individual gram-negative and gram-positive bacterial cells[END_REF][START_REF] Vadillo-Rodriguez | Viscoelasticity of the bacterial cell envelope[END_REF]. On the other hand, the cells are considered as homogeneous materials in order to apply the elastic models. In reality the mechanical response comes from different cellular components depending on the penetration depth.

Results and discussion

Study of the whole PaR3 cells

Morphology

The whole cells morphology was explored by FV, QI and PF-QNM modes, with a scan size set to 3.5 2 µm 2 (Fig. 2). The bacteria diameter for a large majority of cells in a population varies between 350 and 650 nm. This diameter is defined as the height of adherent cell. The diameter determined as the width of the cell is about two times larger. But the latter way of diameter estimation is wrong following the next two reasons.

First, finite tip size causes overestimation of the cell width (geometric artefact). Second, the cells are adherent to the substrate, so their width is increased. The latter fact means as well that the bacteria height is underestimated. Thus, the PaR3 cells diameter from the AFM measurements is 350-650 nm, being a bit underestimated compared to the free non deformed cells. According to the literature, the diameter of Pa cells is 0.5 -1 µm [START_REF] Graham | Nano and microscale topographies for the prevention of bacterial surface fouling[END_REF]. In order to measure the cells diameter by AFM more properly, other technique of cells immobilization should be used instead of electrostatic one. For example, mechanical immobilization in pores of polycarbonate membranes [START_REF] Dague | Towards a nanoscale view of fungal surfaces[END_REF] or holes of PDMS stamp [START_REF] Dague | Assembly of live micro-organisms on microstructured PDMS stamps by convective/capillary deposition for AFM bio-experiments[END_REF][START_REF] Formosa | Generation of living cell arrays for atomic force microscopy studies[END_REF] could be used. However, the non-spherical form of PaR3 cells may provide problems for this latter mechanical immobilization. Coming back to our results, all three AFM modes give the same bacteria height. The height variations for different measurements are only related to variations in the cells morphology.

The repetitive measurements did not display significant changes on bacteria surface, thus the plastic deformation (that is remaining or irreversible deformation) does not play an important role.

Elastic properties

The elastic modulus maps of the PaR3 cells were also obtained for all three FV, QI and PF-QNM modes (Fig. 3). The modulus varies across the cell, increasing towards the center of the cell. This observation is rather interesting. First, it means that there is no substrate effect, otherwise the modulus would be higher towards the cell periphery, where the sample thickness is lower. Second, the bacterium cell does not contain hard organelles, which could cause modulus increase in the specific zone of the cell [START_REF] Longo | Force volume and stiffness tomography investigation on the dynamics of stiff material under bacterial membranes[END_REF]. So, this increase is probably related to geometric artefacts. At the point of maximum cell height the contact between the tip and the surface satisfies the most the condition of symmetric form (Hertz and Sneddon models). So the modulus estimation at this point is the most correct. On the contrary, the tip-surface contact becomes asymmetrical towards the cell edge, thus the modulus estimation following Hertz or Sneddon models becomes wrong. On the other hand, the elastic modulus which corresponds to the substrate is very high and cannot be measured correctly with such a flexible cantilever. The modulus increase towards the cells center was demonstrated before for Gram-negative Shewanella putrefaciens bacterial cells [START_REF] Gaboriaud | Spatially resolved force spectroscopy of bacterial surfaces using force-volume imaging[END_REF].

In the same work a large increase of cells stiffness due to the substrate effect was observed, if the indentation depth exceeded around 60% of the cell thickness. Fig. 3 shows as well the typical force curves for the three AFM modes recorded on PaR3 cells. The curves are displayed in two representations. First, the applied force is plotted against the piezoelectric element movement. The latter represents the sum of the tip-sample separation and cantilever deflection. If the deflection is subtracted, the curves in terms of force against separation are obtained, being shown as well. As one may see, the force curves obtained by QI mode contain rather intense noise, but the latter is easily removed during curves correction stages, and is not disturbing for the fit by a geometric model.

Effect of the scanning conditions on the elastic modulus of PaR3 cells

Effect of the penetration depth

The FV force curves obtained for small 0.2 2 µm 2 zones in the central part of the bacteria were analysed to extract the modulus variation against the penetration depth. The FV curves were segmented into equal penetration length sections that were each fitted with the Hertz model. Two different applied forces were used to probe the elastic modulus at different depth. Three cells were analysed in every case.

First, MLCT-C cantilevers with nominal spring constant of 10 mN/m were used to apply the maximum force of 1 nN. Such common maximum force yielded the deformation around 40 nm. The resulting force curves were then broken into segments of 10 nm width, that were fitted using Hertz model (Fig. 4a). The elastic modulus decreases against penetration depth for the first 40 nm of indentation. This is a specific behaviour, which visualises the effect of hard cell wall, present in bacteria cells.

To reach deeper inside the cell, we used MLCT-D cantilever with the nominal spring constant of 30 mN/m (with an average value of 35 mN/m). It was then possible to reach a maximum force of 8 nN resulting in a penetration depth comparable to the bacterium diameter (Fig 4b). For these high indentations, the elastic modulus increases extremely due to the proximity of the hard substrate beneath the indented cell, or/and to incompressibility of cellular membrane and cytosol. The Hertz model is also used for the modulus estimation. This could seem incorrect, as the deformation is much higher compared to the tip radius. But this consideration has no sense, as the modulus in close proximity of the substrate becomes too high and cannot be measured with such a flexible cantilever. In addition the elastic response becomes extremely non linear, preventing correct application of Hertz model, which suggests the sample homogeneity. Thus the extreme modulus increase cannot be quantified in our test. Interestingly, for PDMS films (relatively soft material) the pronounced substrate effect takes place at deformation corresponding to 45% of sample thickness [START_REF] Xu | Extreme hardening of PDMS thin films due to high compressive strain and confined thickness[END_REF]. As we see from our measurements, for living cells due to their flexibility in changing the shape under applied stress, the substrate effect combined with the effect of cellular material incompressibility happens at much later stage. Another interesting observation is that even totally compressed by the AFM tip, PaR3 cells preserve their viability at least at the timescale of the experiment, as the mechanical behaviour in the end of the scan is the same as in the beginning. Such a high resistance against mechanical compression have been shown already for Gram negative Salmonella typhimurium bacteria cells [START_REF] Suo | Bacteria survive multiple puncturings of their cell walls[END_REF]. Moreover, in the latter study the cells were punctured by an AFM tip when the latter passed through the membrane, as indicated by a specific peak on the approach part of the force curves. Nevertheless the cells stayed alive due to a selfrepairing of lipid bilayers and the peptidoglycan layer. In our case the membrane is not punctured, as no specific peak at approach force curve is present. In the same time, the authors also observed large modulus increase when the tip penetration was comparable to the cells diameter.

The present determination of modulus variation against penetration depth allows defining the large deformation region with rather linear elastic response. The applied force should correspond to this deformation region, if the whole cell is supposed to be tested, with the cytoplasm deformation playing the main role.

Effect of the tip oscillation frequency

The FV mode is usually applied at rather low oscillation frequency <10 Hz. In this work the following question was asked: could the FV mode provide proper force curves with correct modulus values at much higher frequency? Thus the FV mapping was performed with both Bioscope Catalyst and Nanowizard III for frequencies ranging from 3 Hz to 78 Hz for small 0.2 2 µm 2 zones, and the corresponding modulus values were extracted (Fig. 5a-b). All measurements were performed on three different cells for each of three different bacteria preparations to ensure statistically relevant data.

In Fig. 5a and Fig. 5b we compare the FV mode between the JPK Nanowizard III and the Bruker Bioscope Catalyst. In the JPK AFM case, the elastic modulus increases against oscillation frequency, and the dependence is typical for the viscoelastic behaviour (Fig. 5a) [START_REF] Costa | Single-cell elastography: probing for disease with the atomic force microscope[END_REF][START_REF] Guilak | The deformation behavior and viscoelastic properties of chondrocytes in articular cartilage[END_REF][START_REF] Huang | Cell mechanics and mechanotransduction: pathways, probes, and physiology[END_REF].

In the case of the Bruker AFM, the FV measurements provide proper modulus vs. frequency dependence in the low frequency range, while in the range 13-19 Hz the elastic modulus begins to decrease, displaying an abnormal behaviour (Fig. 5b).

Figures 5c and5d present the comparison between both high-speed modes: the QI mode from the JPK AFM and the PF-QNM mode from the Bruker AFM. Again, three PaR3 cells coming from each of three different cultures were analyzed. The modulus vs. frequency dependence from the QI mode (Fig. 5c) displays the expected viscoelastic behaviour of PaR3 cells. These results are consistent with those found in the FV mode. On the contrary, the modulus values obtained by the PF-QNM mode were very low and further decreased with the frequency (Fig. 5d). This tendency for the PF-QNM mode seems to be a continuation of the tendency already observed for the FV mode on the Bioscope Catalyst. The decrease in elastic modulus against frequency for biological sample has already been reported by [START_REF] Dokukin | High-resolution high-speed dynamic mechanical spectroscopy of cells and other soft materials with the help of atomic force microscopy[END_REF]. They attributed this tendency to the creep relaxation. The latter consists in the fact that cell membrane needs some time to establish a stable contact with the tip, so if the latter oscillates too quickly, the contact area is decreased. So, at constant force the stress and consequently the deformation are increased, and the calculated modulus decreases. One may suppose that if the scan area is very small and neighbour sites of tip-surface contacts are very close, thus the cell surface is not fully relaxed after previous deformation, hence preventing an establishment of the symmetric contact after small lateral displacement of the tip. As a consequence, the real contact area decreases resulting in an underestimated elastic modulus. This mechanism may explain the PF-QNM mechanical properties results for the small scanned area. In other words, in the latter case a small surface area is overcharged by very frequent tapping and the surface has not enough time to recover; as a consequence, the symmetric tip-surface contact is not established after tip lateral displacement, leading to the decrease of the contact area and a lower measured elastic modulus. As we have verified, this behaviour is conserved for the 0.5 2 µm 2 central zones (nearly the maximum possible scanning size within a PaR3 cell). In the same time, the correct mapping of the whole bacteria by PF-QNM mode is possible (Fig. 3c).

On the other hand, the operating of FV on the Bruker's and JPK's instruments seems to be different, as for the JPK's AFM the creep relaxation does not take place at the frequency, at which this phenomenon probably takes place on Bruker's AFM. Nanowizard III and Bioscope Catalyst. 3.3.1. On PaR3 cells To explore in more details the difference in modulus variation at high frequency by FV mode on JPK's and Bruker's instruments, the shape of individual force curves was considered (Fig. 6a-b). At the highest explored frequency of 78 Hz the force curves recorded with the Nanowizard III still preserve their shape, with well defined indentation region on both approach and retract parts of the curves (Fig. 6a). We were not able to obtain such proper curves at 78 Hz with the Bioscope Catalyst (Fig. 6b). Indeed, the approach curve still displays an indentation region, but this region becomes rather flat, thus the calculated elastic modulus is very low. As for the retract curve, any calculations of elastic modulus, adhesion or dissipation values would be impossible, as the curve has completely lost its proper shape.

Force curves by FV mode at elevated frequency at

On PDMS standard sample

To confirm a general character of the found FV mode features on JPK's and Bruker's instruments, a standard homogeneous PDMS sample was explored as well in the PBS medium (Fig. 6c-d). The PDMS is rather soft material (elastic modulus around several MPa depending on preparation), nevertheless it is too hard to be tested by MLCT-C cantilevers. Thus only cantilevers were tested in this experiment. The FV scan at 78 Hz on the Nanowizard III has provided again proper force curves with an indentation region available for the fitting process (Fig. 6c). It is interesting to note the presence of small adhesion, which is absent in the case of PaR3 cells. A non-zero adhesion on a hydrophobic PDMS sample means that although the AFM tip is hydrophilic, containing ≡SiOH surface groups, but its hydrophilic character is less pronounced compared to the water. So in the aqueous medium the non modified AFM tip is effectively hydrophobic with respect to the PDMS.

As for the FV scan on the Bioscope Catalyst, the frequency 78 Hz does not allows to get any force curves.

Thus the oscillation at 39 Hz was applied to collect force curves (Fig. 6d). The approach part at this frequency still conserves a proper form, while the retract part of force curves does not present any defined indentation region. In addition, the adhesion force is abnormally high, being non realistic for the contact between the hydrophobic PDMS surface and the polar surface of AFM tip.

Conclusions

The present work aimed to explore the nanomechanical behaviour of living bacteria at high indentation frequencies by applying different AFM modes. We used the novel high-speed multiparametric QI and PF-QNM AFM modes, as well as the classical FV mapping. For small scan area (<0.5 2 µm 2 ) within bacterial cells we found that: i) elastic models remain relevant for indentation frequencies below 20 Hz; ii) above 20 Hz, FV and QI from JPK display the expected increase of Young modulus versus frequency, iii) while FV and PF-QNM from Bruker demonstrated a paradoxical behavior, showing decrease of the Young modulus that we attribute to creep relaxation.

All three FV, QI and PF-QNM modes provide consistent morphology and elastic modulus maps of the whole bacteria. Thus, one may benefit from lower acquisition time and higher resolution by using the QI or PF-QNM modes.

In order to optimize the scanning conditions, an effect of penetration depth on the modulus values was studied using the FV mode. Too low penetration depth (< 20 nm) leads to an increase of measured modulus, as the hard cell wall is tested in such a case. On the other hand, if the penetration depth becomes comparable to the cell diameter, the elastic modulus increases dramatically due to the hard substrate effect and incompressibility of cellular membrane and cytosol.

To establish the tip frequency range appropriate to obtain analyzable results, the elastic modulus behaviour versus the tip frequency was studied by FV, QI and PF-QNM modes for a small central area of the PaR3 cells. The JPK's FV and QI modes displayed the expected viscoelastic behaviour of the cells at different tip frequencies. In particular, the JPK's FV mode could be used at frequencies at least up to 79 Hz, which ensures much lower acquisition time (<1 min) compared to the typical one for this mode (5-10 min). At high tip frequency Bruker's FV and PF-QNM display a paradoxical decrease of the Young modulus that could be attributed to creep relaxation. These findings are also supported by the shape of the force curves on PaR3 cells, as well as on standard PDMS sample. The curves remain appropriate at high frequency by JPK's modes, preserving an indentation region for the fit. At the same time, PF-QNM is well suited to large scan area at the whole cell scale, thus avoiding possible sample overcharging.

Fig. 1 .

 1 Fig. 1. (a) Approach force curve used to calculate deformation and elastic modulus; (b) schematic

Fig. 2 .

 2 Fig. 2. Height images with corresponding profiles for PaR3 in physiological buffer by FV (32 2 px 2 resolution) (a), QI (128 2 px 2 resolution) (b) and PF-QNM (128 2 px 2 resolution) (c) modes. The x and y scales are the same

Fig. 3 .

 3 Fig. 3. Nanomechanical properties of PaR3 by FV (a), QI (b) and PF-QNM (c) modes. From left to right: elastic modulus map with resolution 32 2 px 2 for FV and 128 2 px 2 for QI and PF-QNM; corresponding profile,

Fig. 4 .

 4 Fig. 4. Effect of the indentation depth on elastic modulus by FV mode for three different PaR3 cells

Fig. 5 .

 5 Fig. 5. Effect of the tip oscillation frequency on elastic modulus by different modes: FV on Nanowizard III

Fig. 6 .

 6 Fig. 6. Force curves at elevated frequency by FV mode. (a) PaR3 at Nanowizard III at 78 Hz; (b) PaR3 at

Acknowledgement

This work was supported by the "Fondation pour la Recherche Médicale" (grant ING21040129094). E.D. is a researcher at Centre National de la Recherche Scientifique (CNRS).