Jonathan Tzadikov

Mahmud Auinat

Dr Jesús Barrio

Dr Michael Volokh

Guiming Peng

Christel Gervais

Yair Ein-Eli

Menny Shalom
email: mennysh@bgu.ac.il

Jesfflsb Arrio

Michaelv Olokh

Prof G P M Eng

Shalom

Dr M A Uinat

Prof. Y Ein-Eli

Layered Boron-Nitrogen-Carbon-Oxygen Materials with Tunable Composition as Lithium-Ion Battery Anodes

Keywords: boron-nitrogen-carbon, Li-ion batteries, hydrocarbons, electrochemistry, synthesis design

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

Introduction

Carbon materials have been the focus of immense research due to their unique conductivity and electronic properties. [1,2] The introductiono fh eteroatoms with different electronegativity,r elative to carbon, such as boron, nitrogen, and oxygen, into ac arbon materialc an significantly alter the optical and electronic properties, as well as sensing and catalytic activity. [3][4][5][6][7][8] Therefore, boron-carbon-nitrogen-oxygen (BNCO)m aterials are considered to be very promising materials for various applications, such as electronic, optical, and electrochemical devices,o wing to their tunable opticala nd electronic properties, which depend only on the amount and spatialo rganization of heteroatoms. Traditional BNCO synthetic methodsi nclude solid-state reactions, [9] chemical vapor deposition (CVD), [10] and organic addition reactions of BN to polycyclic aromatic hydrocarbons (PAHs). [11] Although an impressively wide range of materials were synthesized by these methods, there are still some drawbacks that limit furtherp rogress in this field. The CVD methodo ffers low scalability,w hereas the solid-state reactionp rovides moderate control over the composition and positionofheteroatoms within the carbon network and usually includes templating, [12] externalm edia, [13] and condensation of different solid powders. [14] Because the reactive interface of solids is their surface, fine-tuning the elemental composition within aB NCO composite is limited to the surface of the solid reactants. In addition, the reactivity of monomers is impeded by the difficulty of achieving their homogeneous distribution. Unlike solid-state reactions, in al iquid,t he miscibility of reactants ensures both the homogeneity andh igh reactivity of monomers;t huse nhancing control over the synthetic intermediates and final products. We envision that al iquid-state stage spanning aw ide temperature range, before the solidstate reaction, will circumvent many of the solid-state reaction limitations. This methodm ight pave the wayf or the synthesis of new BNCO and other materials with desirable electronic and chemicalproperties and controllable elemental composition.

Herein, we report an ew and straightforward approach to synthesize BNCO materials with controllable elemental ratio and tunable optical and electronic properties by using PAHs, rangingf rom naphthalene( Naph) to fluoranthene (Flu), and ammonia-borane (AB) as startingm onomers. Upon calcination at high temperatures, the two monomers form ac lear liquid solution up to about4 00 8C, before their final condensation into BNCO materials. Ad etailed structural analysis reveals that the monomers react in the liquid phase and establish boronnitrogen-carbon (BNC) bonds. The final BNC composition and BNCO photophysical properties can be easily altered by the The insertion of heteroatomsw ith differente lectronegativity into carbon materials can tune their chemical, electronic, and opticalp roperties. However,i nt raditional solid-states ynthesis, it is challenging to control the reactivity of monomers, and therefore, the amount and position of heteroatoms in the final materials. Herein, as imple, scalable, and general molten-state route to synthesize boron-nitrogen-carbon-oxygen (BNCO) materials with tunable boron-nitrogen-carbon composition, as well as electronic and opticalp roperties, is reported. The new synthetic approachc onsists of polycyclic aromatic hydrocarbons (PAHs) and ammonia-borane as reactants that form a clear liquid-state stage spanning aw ide temperature range, before the solid-state reaction. The molten-state stage enhances the control over the synthetic intermediates and final materials,o wing to improved monomer miscibility and reactivity. The BNCO composition and optical properties are tuned by the PAHs election and final reactiont emperature. The advantages of this method are demonstrated herein through the tunable optical properties, excellent stability to oxidization, facile deposition on substrates, and good activity as an anode material in lithium-ion batteries.

Results and Discussion

BNCO materials were prepared by mixingA Ba nd pyrene (Pyr) at am olarr atio of 1:1. The BNPyr x (x = final condensation temperature) materials were synthesized by placing the monomers in ac eramic crucible and heating to varying temperatures under aN 2 atmosphere (Scheme 1). Upon heating,t he two monomers exhibit am elting point in the range of 100-150 8C. To gether,t he molten monomers are miscible and, above 150 8C, am ilky homogeneous liquid is formed (Figure S1 in the Supporting Information).

XRD patterns (Figure S2 ai nt he Supporting Information) show that below 350 8Ct he monomers does not fully react and mainly patterns of the startingr eactants are detected. For BNPyr 350-700 ,t he XRD patterns imply the formation of layered carbon-like materials. The wide peak at around 22.18 for BNPyr 350 corresponds to an interplanars tacking of BN-containing polymeric materials [15] and amorphous graphite. [16] This peak is shiftedt oh igher values, reaching 24.28 in BNPyr 700 , which indicates as maller distance between the layers in the layered structure as the synthetic temperature rises. FTIR spectra (Figure S2 bi nt he Supporting Information) show ac ombination of characteristic BNCO composite bonds, which are present in all synthesized materials. The weak band at n ˜= 1406 cm À1 is attributed to the BÀNs tretching mode and the intense band at n ˜= 782 cm À1 is associated with BÀNÀBb ending. [17] The bands at n ˜= 1020 and 1083 cm À1 are relatedt oN À BÀOa nd BÀOÀBb onds, respectively, [18] whereas the one at n ˜= 1325 cm À1 corresponds to BÀO. [19] In addition, the presence of aC ÀNb ond is apparent from the band at n ˜= 1235 cm À1 . [20] The band at n ˜% 1577 cm À1 indicates C=Cb onds. [21] It seems that the binding of aB Nu nit to carbon occurs mainly through No rOa toms.

As the final condensation temperature rises above 350 8C, new BNCO materials form with different morphologies and properties to those of the starting monomers. The Raman spectrum (Figure S2 ci nt he Supporting Information) of BNPyr 700 shows Da nd Gb ands at 1340 and 1600 cm À1 ,r espectively.T he Ga nd Db ands are attributed to typical graphitic [22] and defects bands, respectively. [23,24] SEM images of BNPyr x at different calcination temperatures (Figure 1a-d andFigures S3 andS4 in the Supporting Information) indicatet hat the new BNCO materials show ar elatively consistentm orphology,o wing to the homogeneous molten phase as an intermediate. Moreover, the materials are composed of spherical particles, ranging from about 15 to 40 nm, due to the formation of ac olloidal suspensiona th igh temperature andt he boiling of monomers, which possibly results in escapingb ubbles. TEM images (Figure 1e-h and FiguresS5 and S6 in the Supporting Information) reveal that at lower temperatures there is ab lend of thin spherical BNCO particles and some small sheets.A st he condensation temperature increases, the BNCO exhibits al ayered structure and the morphologyo fl arge sheets (> 1 mm) is observed. EFTEM images (Figure 1i-l) of BNPyr 700 show that all elements are uniformly distributed throughout the BNCO material;t his demonstrates that the moltenr eaction forms ah omogeneous material.

X-ray photoelectron spectroscopy (XPS;F igure 2a-d and Figure S7 in the Supporting Information), thermogravimetric analysis( TGA;F igure 2f), and 11 B, 13 CC PM AS NMR spectroscopy (Figure 3) measurements at different reaction temperatures were acquired to elucidate the reaction path, intermediates, and chemical states of all elements in the final materials.

The B1 sX PS spectra (Figure 2a)o fB NPyr 350 show three peaks at 190. 7, 192.7, and 193.9 eV,c orresponding to BÀN, [25] BNÀC, [14] andb orate, [26] respectively.T hese peaks are presenti n other BNPyr samples as well (Figure S7 in the Supporting Information). All peaks mentioned are slightly shiftedt oh igher binding energiesi nB NPyr 700 duet ot he partial oxidation of nitrogen. The C1ss pectra of all BNPyr (Figure 2b and Figure S7 in the Supporting Information) reveal similar existences of sp 2carbon,C ÀN/CÀO, and C=Oa tb inding energies of 284.6-284.9, [27] 286.1-286.4, [12] and 287.3-287.8 eV, [28] respectively.W e could observe only an egligible amount (Figure S7 in the Supporting Information) of CÀBspecies in BNPyr 500 because this bond formation is less favorable energetically. This strengthens the assumption that boron binding occurs mainly through nitrogen and oxygen atoms.

The N1sspectra (Figure 2c and Figure S7 in the Supporting Information) for all BNPyr materials were deconvoluted into three peaks centered at 398.2-398.4, 399.0-399.5, and 400.0-400.2 eV,which were associated with NÀB, [14] NÀC, [29] and amine groups, [27] respectively. For BNPyr 700 ,the amine peak is positivity shifted to 401.7 eV,which indicates the formation of protonated nitrogen groups. [27] Anew peak at 404.4 eV corresponds to either shake-up [29] or oxidized nitrogen [30] .The presence of the abovementioned oxides isconfirmed by O1sXPS measurements (Figure 2d and Figure S7 in the Supporting Information). Postsynthetic oxidation of BCN materials, owing to their low crystallinity, is well described in literature. [31] EELS results for BNPyr 700 (Figure 2e) reveal typical B(188 eV), N (400 eV), C(284 eV), and O(530 eV) Kedges. All atoms show a doublet of p*and s*, which indicates acovalently bonded sp 2hybridized system of BNCO. [6] The growth mechanism and reaction steps of the BNCO materials were studied by TGA of AB mixed with Pyr (Figure 2f) under N 2 .The reaction can be divided into three main stages. First, from 25 to 147 8C, there is asmall weight loss, which corresponds to hydrogen release from the AB monomer and moisture evaporation. [33] The second weight loss, from 148 to 204 8C, the point at which both monomers melt and form a 3 3 stable solution,c an be attributedt ot he releaseo fg aseous molecular aminoborane. [33] The most prominentw eightl oss (69 %) occurs between 205 and 401 8C, with am aximum at 320 8C, at which the monomers react with one other.D uring condensation, borazine andh ydrogen are released, [33] and some unreacted monomers are evaporated.

This reaction temperature is typical for the condensation of carbon materials. The reaction can be easily scaled up, as exemplified hereb yas ingle synthesis of 2g of BNCO material (Figure S8 in the Supporting Information). It is important to note that the pyrolysis of Pyr solely resultsi ni ts full evaporation and decomposition, and no materials were observed afterwards (Figure S9 in the Supporting Information). AB condenses into white BN-likem aterials at high temperature, as previously reported (Figure S10 in the SupportingInformation). [33] This further accentuates the advantage of the liquid phase, which encourages interactions between the two monomers.

The atomic composition of the materials was thoroughly studied by combining elemental analysis (EA) and inductively coupled plasma (ICP) measurements (Table S1 in the Supporting Information). These measurements clearly affirm the successfuls ynthesis of BNCO materials and as uccessful incorporation of large amountso fb oron, nitrogen, carbon, and oxygen. The amounto fc arbon changes from 78.8 wt %f or BNPyr 300 to 48.9 wt %f or BNPyr 700 .U pon condensation at 500 8C, the amount of boron rises, reaching 29.9 wt %. Once the condensation temperature reaches 700 8C, the amount of boron stabilizes at around1 0wt%.T he starting amount of oxygen is 1.5 wt %, whereas for BNPyr 700 it reachesa sh igha s2 4.0 wt %. The amountso fn itrogen are slightly lower than that of boron, due to the releaseo fN H during the reaction.I na ddition, the EA and ICP data suggestt hat the empirical chemical formula is B 1.1 N 1 C 4.4 O 1.7 ,w hich indicates that three AB units react per Pyr molecule.

Further structural elucidation is provided by solid-state NMR spectroscopy data at different calcination temperatures;t his type of spectroscopy is well suited to the structural characterization of BNCO materials. [31] In close agreement with XPS analysis, 11 BM AS NMR spectra show three main signals at all condensation temperatures:as ignal at d iso = 17.6 ppm (C Q = 2.49 MHz, h = 0.2), corresponding to trigonal sp 2 -BO 3 ; [34] a signal at d iso = 0.2 ppm (no quadrupolar shape) duet ot etragonal BO 4 ; [35] and as ignal at d iso = 28.2 ppm (C Q = 3.0 MHz, h = 0.2), indicating the presence of planarB N, [36] whichs uggests domainso fs ix-membered BN rings. BNH 2 environments [37] (d iso = 31.1 ppm, C Q = 3.0 MHz, h = 0.2) are also presenti n BNPyr 300 ,b ut disappear at higher calcination temperatures following the release of hydrogena nd some amine groups. 13 C CP MAS NMR spectroscopy measurements of the materials at different temperatures revealt hat the chemical environment of the graphitic layer is similart ot hat of Pyr units (Figure 3b). At 700 8C, the 13 CNMR signal centered at d = 125 ppm is characteristic of turbostratic graphite, possibly oxidized.

Having all data at hand, we propose the growth mechanism illustrated in Scheme 1a nd SchemeS1i nt he Supporting Information. Upon heating, the two monomers create ah omogeneous ands table liquid solution. Contraryt ot he traditional solid-state reaction, the liquid phase allows the facile organicchemistry-like reaction of the two monomers up to 400 8C. Our results indicate that the formation of BNCOm aterials occurs through the formation of carbon-nitrogen and carbon-oxygen bonds, which meanst hat the AB monomer attacks Pyr.A t higher temperatures, full condensation to ag raphitic carbon with BNO domains is achieved. The similarity of the carbon XPS and 13 CNMR spectra of untreated Pyr and the BNCO materials after calcination at elevated temperatures suggests that the products are composed of many Pyr-likeunits.

The insertion of BN speciesi nto the carbon network leads to materials ranging from semiconductors with tunable absorption (Figure 4a)t os emimetals. [38] The simple alteration of their opticala nd electronic properties through this synthetic methodo pens up av ariety of possibilities towardt heir exploitation in various applications.

The optical properties weree asily tuned by increasing the condensation temperature. UV/Vis spectra (Figure 4a)o f BNPyr x show ar edshift in the absorption up to l = 800 nm with increasing condensation temperature, due to better carbonization of the materials andi mproved packing between the layers. The normalized PL spectra upon excitation at l = 365 nm are given in Figure 4b.I na ccordance with the absorption spectra,aclear redshift of the emission for the materials synthesized at highert emperature was obtained (Figure 4b shows the PL spectra of different powder samples excited at l = 365 nm). Interestingly,t he PL spectrum of BNPyr 500 is broader and coversa lmosta ll of the visible region;t his may be attributed to an ew electronic transition, owing to the formation of more defects sites on the surfaceo ft he material with increasing condensation temperature. [39] Notably,r elative to BNPyr materials, the emission spectrum of Pyr (Figure 4b)i s different andblueshifted. Furthermore, the BNCOs can be exfoliated by sonication in 2-propanol, resultingi nastable colloidal suspension with strong and tailoredo pticalp roperties (Figure S11i nt he Supporting Information). The PL spectra of BNCO dispersions are blueshifted relative to the corresponding powders, owing to successful exfoliation of the layers.

To demonstratet he generality of this method, we utilized AB with four other commonP AHs, namely,N aph, Ant, Phe, and Flu, as monomers instead of Pyr.T he full characterization of all materials at different calcination temperaturesi sg iven in Figures S12 andS 13 in the Supporting Information. The other PAHs melt at relativity similart emperatures and establish ah omogeneous solution with AB. Similar to Pyr,t he new PAHs react with AB to form BNCO materials with different photophysicalp roperties, ranging from semiconductors to metals (Figure 4e-f).

EA data (Figure 4d and Table S2 in the SupportingI nformation) shows that the carbon content within the material changes according to the starting PAHs;t hus, the selection of the PAHa llows the possibility to tune the final BNCO photo-physicala nd chemical properties. Furthermore, there is ad irect correlation between the startingP AH unit and the final optical properties, for example, the BNCO absorption edge ranges from near-UVw ith Naph to the visible region with Flu. This trend is confirmed by PL measurements (Figure 4e), for which BNNaph 350 exhibits an emission at l = 420 nm, whereas the emission of BNFlu 350 is redshifted to l = 530 nm.

Another important advantage of this synthetic route lies in its straightforward growth and deposition on substrates, such as FTO. Usually,s trong van der Waals interactions in layered materials (e.g.,g raphitic carbon nitride, boron nitride) and their low solubility make it almost impossible to deposit them on substrates. The molten-phase stage allows the formationo f an intimate contact with the substrate. Upon condensation, followed by cooling to room temperature, au niform layer with tunable optical properties can be acquired. As ap roof of concept, we deposited BNCO materials by using the same synthetic approach and placing FTO glass below the starting powder.

Figure 4c shows images of BNCO materials on FTO glass that werep repared from BNPyr and BNPhe at 350 and 400 8C. SEM images (Figures S15 and S16 in the Supporting Information) confirm the successful deposition of BNCO on FTO glass withoutp inholes.T he UV/Vis absorption edge (Figure 4c)o f the covered FTOs is blueshifted compared with the correspondingp owder samples, owing to thinner layer deposition. The thin layer also explains the similarity between the XRD patterns of the covered FTOs (Figure S17 in the Supporting Information)a nd plain FTO. FTIR spectra (Figure S17 in the Supporting Information) shows bands identical to that of BNCO powder. The facile growth of BNCO materials, together with their intriguing photophysical properties, encourage their utilization in photoelectronic devices, such as solar cells, photoelectrochemical cells, and light-emitting diodes.

The insertiono fh eteroatoms into ac arbon matrix usually increasesi ts stability to thermal oxidation. [5] TGA analysis (Figure S18 in the Supporting Information) shows that BNCO 700 exhibits superior stability to oxidation and about 80 %o ft he startingm aterials is preserved upon heating to 1000 8Ci na ir, which indicates that the materials can be used for applications that require high stability to oxidation.

Boron, due to its slightly lower electronegativity than that of carbon,i nduces electron-rich carbon, whereas nitrogen insertion causes surface defects that result in more reactive sites, [40] which should be beneficial for lithium-ion intercalation. It has been shown that BCN materials have relatively good capacity values when acting as an anode in al ithium-ionc ell setup. [41,[START_REF]Nanotubes and Nanosheets-Functionalization and Applications of Boron Nitride and OtherM aterials[END_REF] Therefore, we preformed preliminary studies on the utilization of the synthesized BNCO materials for reversible lithium-ion hosting. For this purpose, charge-discharge characteristics of the materials in ah alf-cell configuration versus lithium metal were evaluated, and the capacity and cycle stability of the BNCOs were deduced.F ives amples were synthesized at 700 8C with AB/Pyr molar ratios of 5:1, 2:1, 1:1, 1:2, and 1:5t od etermine the optimal composition. FTIR, XRD, EA, and ICP measurements are shown in Figure S19 and Ta ble S3 in the Supporting Information. Notably,a th igher calcination temperatures, the activity of the lithium-ion batteries decreases due to an increasedn umber of defects in the structure (Figures S20-23 and Ta bleS4i nt he Supporting Information).

The electronic conductivities of samples with AB/Pyr molar ratios of 5:1, 1:1, and 1:5a re 0.01, 0.50, and 0.65 mScm À1 ,r espectively;t his shows ag ood correlation between the starting amount of Pyr and C/N ratio (Table S5 in the Supporting Information). [START_REF] Dong | [END_REF] Figure5 presents the charge-discharge profiles of cycles 1, 5, 10, 50, and 100 of the 1:1B NPyr 700 composite electrode material, which were recorded during polarization at a constantc urrent of 0.1 mA cm À2 in ah alf-cell configuration versus Li metal (Figure 5a), and the cycle life stability analysis of the same cell (Figure 5b). The recorded capacity values are comparable to that of state-of-the-art graphite-based anodes. [44] Figure S24 in the Supporting Information presents the life cycle stabilityo ft he abovementioned materials with different AB/Pyr ratios, and it is evidentt hat the ratio between the starting materials has ad rastic influence on the measured capacity. Whereasalow Pyr content in the synthesis results in rather low reversible capacity values, the stability of all BNCO materials upon cycling is good. This is strongly evident in Figure 5c, which describest he cells being cycled at different current densities, from 0.1 to 1.6 mA cm À2 in ar eversible staircase mode. Whereast he initial capacity recordeda tacurrent density of 0.1 mA cm À2 is 350 mA hg À1 ,i ti sr educed to slightly to less than 300 mA hg À1 if the current density is doubled to 0.2 mA cm À2 .T he materials could sustain high capacity (225 mA hg À1 )i ft he current was doubled again to 0.4 mA cm À2 .I ft he current was drastically increased to 0.8 and 1.6 mA cm À2 ,t he capacities were still impressively high:1 80 and 140 mA hg À1 ,r espectively.I ft he current wasd ecreased back in as taircase mode of operation, the capacities recovered back to their originalv alues. This is as trong indicationo f rather fast lithium kinetics in the materials tructure, while maintaining high gravimetric capacities.

The observed capacity trend is expected because the carbonaceousc haracteristics within the materials might eventually be the factor that determines the reversible capacity.I na ddition, whereas the measured reversible capacity upon cyclingi s increased with increased Pyr content, the irreversible capacity, partially associated with the build-up of ap assivating film, [START_REF] Ein-Eli | .Ageneral scheme for the synthesis of BNCO. Figure 1. SEM images of BNPyr calcineda ta )350, b) 400,c)500, and d) 700 8C. TEM images BNPyr calcinedate )350, f) 400, g) 500, and h) 700 8C. Energy-filtered (EF) TEM imagesofB NPyr 700 :i )boron ([END_REF] is quite high for all electrode compositions. This may be wellc orrelated with the relatively high oxygen content in the BNCOs, as discussed earlier.I ti sp ostulated that, within the first lithiation processes of the BNCOs, lithium oxide species may be formed. [32] In-depth surfacea nalyses are being planned to better understand the performance of the materials. Such an understanding may pave the waytobetter design BNCO materials, as well as tuning the electrolyte composition, enabling a substantial decrease in the irreversible capacities, while maintaining the high stabilityu pon extensive cycling.

Conclusions

We have demonstrated as imple and general molten-state synthesis of BNCO materials with controllable BNC composition and tunable optical, chemical, and electronic properties.T he new synthesis becomes possible by using monomers with a distinct molten-phase window at high temperatures, namely, PAHs and AB. Similart ot he common organic synthesis approach,t he liquid-phase stage endows betterm iscibility and reactivity of the reactants compared with that of traditional solid-state reactions. Detailed structural analysis of the liquidstate intermediates and final BNCOm aterials at different calci-nation temperatures reveals that the reaction occurs through the establishment of aB ÀNÀCb ond, followed by further condensation to BNCO materials. We demonstrated that both the chemicalc omposition ando ptical and electronic properties could be tuned by selecting different PAHs, owing to the different carbon contents,a nd calcination temperatures.T he liquid-state stage allowst he deposition of at hick and uniform BNCO layer on ag iven substrate. The materials exhibit excellent stability to thermal oxidization, tunability of absorption and photoluminescence properties, as wella sg ood activity as an anode materiali nl ithium-ion batteries. We believe that the generality,s implicity,a nd scalability of this synthetic approach opens up opportunities for the synthesis of new materials (BNCO and others) with controllable elemental composition, as well as variable opticala nd electronic properties, toward their utilization in photoelectronic and electrochemical devices.

Experimental Section

Chemicals AB complex (90 %, Sigma-Aldrich, 104 8Cm elting point), Naph (99 %, Acros Organics, 80-82 8Cm elting point), Ant (99 %, Acros Organics, 214-218 8Cm elting point), Phe (98 %, Alfa Aesar,9 7-101 8C melting point), Pyr (98 %, Acros Organics, 145-148 8Cm elting point), and Flu (98 %, Sigma-Aldrich, 105-110 8Cm elting point) were used as received.

Synthesis

To prepare BNCO, Naph, Ant, Phe, Pyr,a nd Flu were mixed with the AB complex at am olar ratio of 1:1. Syntheses were carried out under an inert N 2 atmosphere at varying temperatures:2 50, 300, 350, 400, 500, 700, and 800 8C. The mixtures were heated at ar ate of 2.4 8Cmin À1 and held at the target temperature for 4h.

To deposit BNCOs on FTO substrates, either Pyr or Phe were mixed with AB complex (1:1 molar ratio) inside ac eramic crucible with FTO-covered glass on its bottom, with the conductive side facing up (facing the powder). Syntheses were carried out to different target temperatures ranging from 350 to 400 8C. The crucibles were heated under aN 2 atmosphere at ar ate of 2.4 8Cmin À1 and held at the target temperature for 4h.

Conductivity measurements

BNCO powders were ground by using aF ritsch Pulverisette 7p lanetary ball mill (3 mm ZrO 2 balls, for 45 min). The powders were then vacuum dried and their I-V curves were measured in ac onductivity cell by using atwo electrode setup.

Anodee lectrodep reparation

Preparation of BNCO materials for Li-ion half-cell studies was executed by mixing Pyr with AB at molar ratios of 5:1, 2:1, 1:1, 1:2, and 1:5. The mixtures were heated at ar ate of 2.4 8Cmin À1 and held at the target temperature for 4hunder aN 2 atmosphere. The obtained materials were ground by using an agate mortar and pestle, followed by 325 mesh sieving, to give particles of less than 44 mm. Slurries of the active BNCO materials (90 wt %) were prepared by mixing them with acetylene black (Super-P carbon), fol-lowed by the introduction of the mixture into N-methylpyrrolidinone (NMP) containing dissolved polyvinydene difluoride (PVDF). Electrodes were prepared by doctor blading the slurries on copper foil of 12 mmf ollowed by overnight drying in av acuum oven at 120 8C. The resulting composite BNCO electrodes held (3 AE 0.2) mg cm À2 with ac omposition of BNCO/Super-P/PVDF in ar atio of 8:1:1.

Electrochemical BNCO anode evaluation

Disk-shaped electrodes (with as urface area of 1.13 cm 2 )w ere cut and placed versus lithium-metal disk electrodes in aT -cell shaped electrochemical apparatus, by utilizing ethylene carbonate/dimethyl carbonate (EC/DMC;1 :3 v/v) electrolyte containing 1 m LiPF 6 . These half-cells were studied in ac onstant-current cycling mode of polarization (0.1 mA cm À2 )b etween the potentials of 2.00 and 0.01 Vv ersus Li/Li + .

Characterizationo fm aterials

XRD patterns were obtained by using aP ANalytical Empyrean diffractometer.AJ EOL JSM-7400F high-resolution scanning electron microscope, equipped with aF EG source, operated at 3.5 kV.T EM images were recorded on an FEI Te cnai T-12 G2 TWIN instrument operated at 120 kV.H igh-resolution (HR) TEM imaging, EFTEM, and EELS were carried out on aJ EOL JEM-2100F analytical transmission electron microscope operated at 200 kV equipped with GATAN8 94 US1000 camera. EFTEM experiments were performed by using a Gatan image filter.C arbon (284 eV), oxygen (532 eV), nitrogen (400 eV), and boron (188 eV) Ke dges were used for elemental mapping by means of the three-window method. EELS dispersion: 0.25 eV/ch, aperture 2.5 nm. Scanning TEM (STEM) images were recorded by using aG ATAN 806 HAADF STEM detector.T he probe size during analysis was set to 1nm. FTIR spectroscopy performed on aT hermo Scientific Nicolet iN 10MX IR microscope. Optical absorbance spectra were measured by using aC ary 100 spectrophotometer equipped with an integrating sphere, in reflectance mode. Fluorescence measurements were collected by using aF LS920P spectrofluorimeter.T he XPS data were collected by using an X-ray photoelectron spectrometer (ESCALAB 250) ultrahigh vacuum (1 10 À9 bar) apparatus with an Al Ka X-ray source and am onochromator.T he X-ray beam size was 500 mma nd survey spectra were recorded with ap ass energy (PE) of 150 eV,a nd high-energy-resolution spectra were recorded with aP Eo f2 0eV. To correct for charging effects, all spectra were calibrated relative to the C1 sp eak, positioned at 284.6 eV.T he XPS results were processed by using the AVANTAGE software. Solid-state 11 BM AS and 13 CC PM AS spectra were recorded on aB ruker AVANCE 700 spectrometer (16.3 T, n 0 ( 1 H) = 700.29 MHz, n 0 ( 11 B) = 224.68 MHz, n 0 ( 13 C) = 176.09 MHz) by using a4mm Bruker probe and as pinning frequency of 12 kHz for 13 Ca nd 14 kHz for 11 B. 13 CC PM AS experiments were recorded with ramped-amplitude cross polarization in the 1 Hc hannel to transfer magnetization from 1 Ht o 13 C. (Recycle delay = 600 sf or Pyr,3sf or heat-treated samples, CP contact time = 1ms, optimized 1 Hs pinal-64 decoupling.) 11 BM AS NMR spectra were recorded by using a spin-echo q-t-2q pulse sequence with q = 908,t oo vercome problems related to the probe signal. The t delay was synchronized with the spinning frequency.C hemical shift values were referenced to tetramethylsilane for 13 Ca nd BF 3 •OEt 2 for 11 B. Mass loss of the monomers, reaction intermediates, and the final material were obtained in at hermogravimetric analyzer (Q500). EA data for CHNSO was collected by using aT hermo Scientific Flash Smart elemental analyzer (OEA 2000). Boron content was acquired by dissolving the samples in concentrated nitric acid in an autoclave for 12 ha t 180 8C, and analyzing the sample in aS pectro ARCOS ICP-OES, FHX22 MultiView plasma (SOP,E OP), instrument. The Raman system comprised of aH oriba LabRam HR evolution micro-Raman system, equipped with aS ynapse Open Electrode CCD detector air-cooled to À60 8C. The excitation source was a l = 532 nm aircooled frequency doubled Nd:Yag laser.T he laser power on the sample was about 0.5 mW.T he laser was focused with a 100 objective to as pot of about 1 mm. The measurements were taken with a6 00 gmm À1 grating and a1 00 mmc onfocal microscope hole. The typical exposure time was 30 s.

Figure 2 .

 2 Figure 2. XPS spectrao fB NPyr 350, 700 for a) B1s, b) C1s, c) N1s, and d) O1s. e) Electron energy loss spectroscopy (EELS) results for BNPyr 700 ,and f) TGA and derivative weightc urves of the reaction between AB and Pyrunder N 2 .

Figure 3 .

 3 Figure 3. a)11 BMAS NMR spectra of AB and BNPyr 300, 500, 700 .A simulation is proposed as an illustration for BNPyr 300 ;b)13 CCP MAS NMR spectra of Pyr and BNPyr 300, 500, 700 .

Figure 4 .

 4 Figure 4. a) UV/Vis and b) normalized photoluminescence (PL;e x. l = 365 nm) spectraand imagesoft he powders of BNPyr 300, 350, 400, 500 .c)UV/Vis spectra of BNPhe 350, 400 and BNPyr 350, 400 on fluorine-doped tin oxide (FTO), and imageso ft he coveredF TOs. d) CcontentinB NCO [wt %] with different PAHs. e) UV/Vis and f) normalized PL (ex. l = 365 nm) spectra of BNPAH 350, 400 (naphthalene (Naph),a nthracene (Ant), phenanthrene (Phe), and fluoranthene (Flu)). The unnormalized PL spectrum is given in Figure S14 in the Supporting Information.

Figure 5 .

 5 Figure 5. a) Charge-discharge profiles (cycles1 ,5,1 0, 50, and 100) of 1:1 BNPyr 700 obtainedfrom polarization under ac onstantc urrent of 0.1 mA cm À2 in ahalf-cell configuration versus Li metal. b) Life cycle stabilitya nalysis of the samecell. c) Performance rate of BNPyr 700 material:c ells were cycled in a staircase modeo fpolarization,a tdifferentc urrent densities of 0.1-1.6 mA cm À . 2

Acknowledgements

We thank Dr.V ladimir Ezersky,D r. Sofiya Kolusheva,D r. Natalya Froumin, Dr.L eila Zeiri, Dr.A na Millionshchick,a nd Dr.A lexander Upcher from the Ilse Katz Institute for Nanoscale Science and Technology for their help in characterizations. We acknowledge financials upportf rom the Israel Science Foundation( ISF,g rant no. 1161/17), and the Israel NationalR esearch Center for Electro-chemicalPropulsion (INREP).

Conflict of interest

The authors declare no conflict of interest.


Thomas Söllradl

Kevin Chabot

Ulrike Fröhlich

Michael Canva

Paul G Charette

Michel Grandbois
email: michel.grandbois@usherbrooke.ca

Thomas Söllradl Abcd

Kevin Chabot Ab

Monitoring individual cell-signaling activity using combined metal-clad waveguide and surface-enhanced fluorescence imaging

come

Introduction

Eukaryotic cells express a wide variety of structural and functional components to fulfill their tasks and maintain tissue and organ functions. For example, the cytoskeleton of smooth muscle cells generates and maintains forces to regulate vascular tone or engage in rhythmic, synchronized contractions. 1 To sustain individual cell activity associated with physiological functions, cells express a wide variety of membrane-bound receptors that respond to a wide variety of external biochemical stimuli. The largest class of such receptors is the G-protein-coupled receptors (GPCRs), which control a variety physiological processes including cell-cell communication, vascular tone, hormonal signaling, and neuronal transmission. 2 The reliable detection and quantification of GPCR activity is of great interest in the fields of cellular biology and pharmacology.

Cell-based assays using a variety of label-free transduction methods such as diffraction gratings, 3 electrical impedance, [START_REF] Tiruppathi | Proc. Natl. Acad. Sci. U. S. A[END_REF] and surface plasmon resonance [START_REF] Chabot | [END_REF][6][7][8][9][10][11] have been proposed to study cellsignaling with no need for exogenous markers. Recently, a more flexible type of evanescent field transduction based on metal clad waveguides (MCWG) has been used in sensing applications with cells and bacteria to probe deeper into the cell body 12,13 than is possible with SPR (typically 200 nm or less for visible and near-IR systems). Using appropriate optics, SPR and MCWG can be used for imaging where advanced systems are capable of resolving intracellular structures. Our group and others have demonstrated this capability in a variety of cell-substrate interaction experiments, 8,[14][15][16] in particular to provide insight into the heterogeneous response of a cell population undergoing apoptosis. 14 Note that spatial resolution in SPR and MCWG images is limited along the direction of light propagation by the finite mode attenuation length, rather than by diffraction. 17,18 Furthermore, there is an intrinsic trade-off between mode attenuation length and penetration depth into the sensing medium such that any increase in penetration depth comes at the expense of a commensurate decrease in imaging spatial resolution. Compared to SPR, MCWG offers the better compromise between spatial resolution and penetration depth. 13 Hence, when imaging relatively thick objects such as eukaryotic cells or bacteria, this tradeoff is especially relevant and MCWG-based imaging is preferred.

Since SPR and MCWG are label-free methods, they yield direct information on local changes in material properties (in this case the refractive index), rather than on specific molecular targets. As with all label-free methods, however, it can sometimes be challenging to unambiguously identify molecular targets or cell structures from the image data. Indeed, though various SPRbased imaging experiments have been applied to study cellular structure and functional activity, few studies have attempted to identify the particular subcellular structures involved. 8,[19][20][21][22] Some of the most successful attempts involve separate corroborating experimental measurements with conventional fluorescence microscopy to identify a-posteriori the cellular features in the labelfree micrographs. 8,19 Image fusion methods attempt to provide the best of both worlds by combining distinct imaging modalities, ideally in a single instrument, such as with simultaneous atomic force and confocal fluorescence microscopy. Evanescent field based methods such as MCWG and SPR are capable of simultaneous label-free and fluorescence imaging, where the evanescent field in the dielectric Version of the laboratory Please find the article on Analyst website -https://doi.org/10.1039/C8AN00911B can be used to excite fluorescent markers near the surface in a manner similar to total internal reflection fluorescence (TIRF) microscopy. Furthermore, the resonant coupling to the guided surface mode in MCWG and SPR produces a significant field enhancement in the sensing medium compared to TIRF, as demonstrated with surface plasmon enhanced fluorescence (SPEF). 23 Indeed, SPEF has been shown to increase detection sensitivity in classic fluorescence-based biomolecular assays by orders of magnitude. 24 More recently, our group and others applied SPEF in cell sensing to identify the specific cellular processes underlying an SPR response. 22,25,26 In this work, we introduce the first microscopy platform to combine MCWG and surface-enhanced fluorescence (SEF) imaging, a method we term "MCWG-SEF microscopy". Relative to SPR, the enhanced probing depth of MCWG enables simultaneous label-free and fluorescently-labeled imaging of cellular structures further up into the cell body, while the improved trade-off between resolution and probing depth maximizes the achievable spatial resolution. A first series of validation experiments are presented where actin stress fibers are simultaneously imaged by MCWG and SEF in vascular smooth muscle cells (VSMC). A second series of experiments is presented showing intracellular signaling associated with the activation of AT1R by its agonist AngII in a stably transfected cell model. Intracellular calcium levels, a robust and specific marker of AT1R signaling, are recorded by SEF with the fluorescent probe Fluo8, while simultaneously capturing the whole cell response with MCWG imaging. Use of a pharmacological inhibitor made it possible to correlate specific downstream signaling pathways and heterogeneity in the AngIIdependent cell response with the information in the MCWG images.

Materials and methods

MCWG-SEF microscopy platform

The MCWG-SEF microscopy platform is an expansion on a custom-built MCWG imaging system based on a high-numerical-aperture objective described in detail elsewhere. 13 The sensor chips (MCWG substrate) with the cells under study are placed in a fluid chamber (QR-43C, Harvard Apparatus). As shown in Fig. 1, the light sources are collimated low-coherence fiber-coupled LEDs (λnom = 470 nm from Thorlabs or λnom = 530 nm from Mightex Systems) chosen to minimize diffraction artifacts and phototoxicity. Input light passes through a bandpass filter (530/10 or 470/20, Chroma Tech) and is focused at the back focal plane of a high-numerical-aperture microscope objective (63×, NA = 1.46, Zeiss). The offset of the focused spot from the objective's optical axis determines the incidence angle of the collimated input light at the backside of the MCWG substrate. The reflected light is collected back through the objective where a polarizing beam-splitter separates each component for visualization by a pair of CCD camera (1392 × 1040, 12 bit, gray-scale, Allied Vision) at an acquisition rate of 1 Hz. As designed, the MCWG sensor chips support a single (fundamental) transverse magnetic (TM) mode and a single (fundamental) transverse electric (TE) mode. The incidence angle of the input light is chosen to couple solely with the TM mode since it provides the best tradeoff between spatial resolution and penetration depth. 13 Thus, only intensity variations in the TM-component of the reflected light contain information regarding refractive index variations in the sample. In general, however, there may also be source-dependent spatial and temporal intensity variations in the TM-based signal. Since the TE-component of the reflected light is totally internally reflected at the glass/metal interface of the MCWG chips and thus does not interact with the sample, it can be used as a reference to Version of the laboratory Please find the article on Analyst website -https://doi.org/10.1039/C8AN00911B correct the TM-based signal for source dependent intensity variations. Fluorescence light is collected simultaneously through the top window of the sample chamber using a long-workingdistance microscope objective (50×, 0.55, Mitutoyo Inc.), passes through an emission filter (HQ525/50 from Chroma Tech or LP615 from Zeiss), and is imaged by a cooled CCD camera (1392 × 1040, 16 bit, Qimaging). Note that while fluorescence imaging is diffraction-limited, spatial resolution in the MCWG images is anisotropic: resolution is limited along the direction normal to light propagation in the image plane by diffraction and by the finite mode attenuation length in direction of propagation. As stated above, MCWG provides a lower level of anisotropy compared to SPR at deeper probing depths. The MCWG substrates are in fact planar optical waveguides fabricated on BK7 microscope slides consisting of a thin metal layer as the bottom cladding, a dielectric core, and a liquid sensing medium with the cells as the topmost cladding. 13 Refractive index variations at the sensor surface Version of the laboratory Please find the article on Analyst website -https://doi.org/10.1039/C8AN00911B caused by changes in cell morphology and/or internal cell reorganizations locally change the guided mode properties, which in turn result in reflected intensity changes recorded by the TMcomponent CCD camera. The relatively large refractive index differences (1.355 < n < 1.60) between the cellular organelles and the cytosol, as well as between the organelles themselves, 27 are easily resolvable in the MCWG images. Finally, the evanescent field in the liquid sensing medium excites fluorophores near the sensor surface, similarly to TIRF microscopy, yielding simultaneous MCWG and SEF imaging.

Cell culture and fluorescence staining

In a first series of experiments, an immortalized rat aortic vascular smooth muscle cell line (A7r5, ATCC, USA) was used to image the actin cytoskeleton components, where the cells were seeded on ethanol-sterilized MCWG substrates and F-actin was labeled with Alexa Fluor 532-phalloidin. Cells were maintained within selective Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% heat-inactivated fetal bovine serum (FBS), 2.5 μg ml -1 amphotericin B, 50 IU ml -1 penicillin, 50 μg ml -1 streptomycin and supplemented with 1% non-essential amino acids. Post-fixation in 4% paraformaldehyde (Sigma-Aldrich) for 10 min, cells were washed twice with phosphate-buffered saline (PBS, Sigma-Aldrich) and incubated in tris-buffered saline (TBS, pH 8.1, Sigma-Aldrich) for 5 min. After additional washing with PBS, the cell membrane was permeabilized using a 0.1% triton solution (Sigma-Aldrich) for 1 min followed by a thorough wash using PBS. Actin was stained by incubating the samples with Alexa Fluor 532-phalloidin (1 : 20 Thermo Fisher Scientific) for 20 min. Samples were then washed and used immediately for imaging.

In a second series of experiments, intracellular GPCR signaling activity was studied in human embryonic kidney 293 cells (HEK 293, Qbiogene, QBI HEK-293A cells, USA), stably transfected to express the AT1R as described previously. 28 Cells were maintained within selective DMEM supplemented with 10% heat-inactivated FBS, 0.4 mg ml -1 G-418, 2.5 μg ml -1 amphotericin B, 50 IU ml -1 penicillin and 50 μg ml -1 streptomycin. To observe intracellular calcium levels, HEK-AT1R cells were seeded at a density of 5.75 × 10 5 on poly-L-lysine-coated sensor chips and grown to 60% confluence. Cells were washed twice with warm Hank's balanced salt solution (HBSS) and incubated with the calcium indicator Fluo-8 (5 μM, Thermo Fisher Scientific) for 1 h at room temperature, washed twice and left for another 30 min in HBSS for de-esterification prior to an experiment. AT1R-activation was performed by exchanging the media in the sample chamber using a syringe pump (100 μl min -1 for 8 min) with HBSS buffer containing 100 nM Angiotensin II (American Peptide). ROCK kinase activity was inhibited by pre-incubating the cells with Y27632 for 30 min (10 μM, Sigma-Aldrich) and the inhibitor was present throughout the experiment.

The microscopy platform relies on two separate optical systems to acquire the MCWG and SEF images from a sample, where the images reflect distinct types of information (MCWG: refractive index variations across the entire field of view, SEF: fluorescence intensity from specifically labeled cellular components). Although the fields of view and pixel correspondence between the two systems could be matched with appropriate optics, the depths of fields for both techniques are quite different and MCWG-based imaging has a characteristic planar spatial resolution imbalance. As a result, even with perfect pixel-correspondence, sub-cellular structural features may be difficult to correlate between the MCWG and SEF images. Indeed, this "correspondence problem" is common to all image-fusion systems, such as with PET (position emission Version of the laboratory Please find the article on Analyst website -https://doi.org/10.1039/C8AN00911B tomography) and CT (X-ray computed tomography) for example. In general, the solution to this problem is to ensure that the samples contain clearly identifiable anatomical features in both images which can be used as spatial references. In the case of sub-cellular resolution imaging, this can be particularly challenging.

Results and discussion

Actin cytoskeleton imaging by MCWG-SEF microscopy

VSMCs exhibit a densely structured network of actin filaments 29 and are the mechanically active cell layer responsible for regulating vascular muscle tone and blood pressure. Fig. 2A shows a series of MCWG micrographs of a single VSMC adhered to the sensor surface taken at increasing angles of incidence (θi) to resonantly couple to a range of refractive indices in the sensing medium. At 64.17° (Fig. 2A), resonant coupling to the fluid medium is achieved (darkest background indicating lowest reflectivity) and evanescent field intensity is maximized, where the dominant features in the MCWG micrograph are filamentous structures aligned with the long cellular axis. The greater probing depth provided by MCWG enables the visualization of structures in these cells that are consistent with actin filaments. 30 To confirm that these structures are indeed actin fibers, the high-affinity F-actin probe phalloidin-Alexa Fluor 532 was used to label the cell cytoskeleton and SEF micrographs were simultaneously recorded (Fig. 2B). As expected, the highest SEF signal intensity is achieved at 64.17°, an angle at which a good correlation between the elongated structures in the MCWG and SEF micrographs is observed. As explained above, spatial resolution in the imaging plane is anisotropic for label-free imaging modality. In our MCWG-SEF microscope, spatial resolution in the MCWG images is limited by diffraction limit of ∼0.18 μm in the direction normal to mode propagation and to ∼5.0 μm in the direction of mode propagation (black arrow in leftmost image in Fig. 2A) due to the finite attenuation length of the mode. 13 This effect can also be seen in the second image from the left in Fig. 2A (64.17°) where the fibers aligned in the direction of propagation are clearly resolved (white arrow) while structures that are slightly off-axis appear partly blurred (white dotted arrow). Note since the fluorescent labels acts as point sources of light, the SEF signal is not subject to this effect (spatial resolution in the SEF images is isotropic and diffraction-limited in both axes). In addition, as observed by others with SPR, 31 the presence of fluorophores with an absorption band that overlaps the excitation light spectrum locally modulates the (complex) refractive index in the sensing medium, acting as a contrast enhancement agent in the MCWG images. Fig. 2C shows simultaneously acquired normalized plots of MCWG reflected light intensity (averaged within the ROI shown by the black square in Fig. 2A at 62.05°) and SEF intensity (overall image average) as a function of incidence angle. The minimum in MCWG reflected light intensity at θi = 64.82° is known as "critical coupling". At this angle, the electric field intensity at the sensor surface is very strong, yielding high fluorescence intensity (grey trace), as previously observed by our group and others for SPR. 13,23 As expected, maximum fluorescence intensity (θi = 64.17°) is slightly shifted from the minimum MCWG reflected light intensity since critical coupling occurs slightly off-resonance, where there is just enough out-coupled light from the guided mode to cancel the un-coupled incident light by destructive interference (in the SPR literature, [START_REF] Raether | Surface plasmons on smooth and rough surfaces and on gratings[END_REF] this is referred to as the internal and re-radiation damping being balanced).

AT1-induced cell activity analyzed by MCWG-SEF microscopy

Cell-based, label-free technologies are now routinely used to monitor cellular responses associated with the activation of signaling pathways in cell populations to provide insight into their complex mechanisms and functions. [START_REF] Schröder | [END_REF]34 While GPCR signaling is often assumed to be homogeneous within a cell population, receptor expression levels and variations in individual cell phenotypes can alter their overall response profile and lead to heterogeneous population responses. 35,36 To demonstrate the possibility of assessing such population-dependent behavior, we used a HEK293 cell model stably transfected with AT1R. 37 In this cell model, two major signaling cascades are involved following receptor activation by AngII, namely the Gq-protein dependent signaling cascade leading to intracellular calcium mobilization and the Version of the laboratory Please find the article on Analyst website -https://doi.org/10.1039/C8AN00911B G12/13/Rho/ROCK pathways. These pathways are known to be involved in both the regulation of the cytoskeleton organization and actin-myosin-dependent contractile activity. 38,39 We used the MCWG-SEF microscopy system to follow intracellular calcium levels with the fluorescent probe Fluo8 (SEF imaging) while simultaneously monitoring the overall cellular morphological response (MCWG imaging) resulting from AT1 receptor activation by AngII. Fig. 3 shows a time series sample of eight individual cells imaged with MCWG (left image column) and SEF (right image column). As expected, the cell morphology in the MCWG and SEF images (Fig. 3A andG) appear slightly different since the MCWG signal in our configuration originates from nonspecific refractive index differences within a ∼0.5 μm thick sensing volume 13 whereas the SEF signal originates from the presence of a calcium indicator distributed within a ∼1 μm thick volume above the sensor surface. MCWG micrographs B-E are color-coded maps of percentage reflectance difference relative to the reference (Fig. 3A), where the blue end of the spectrum indicates a decrease, the red end of the spectrum indicates an increase, and green indicates no change. Following AngII stimulation at 0 min, a burst of intracellular calcium is observed within the first 5 min in the SEF images and quantified in the average fluorescence intensity plots, confirming receptor activation and functionality of the signaling pathway in this cell model. The burst of intracellular calcium is followed by the appearance of localized decreases in MCWG reflectance (blue regions) attributable to a reduction in cellular mass density within the effective sensing range of the evanescent field, consistent with increased acto-myosin contractile activity, 21,28 where the minimum MCWG average signal intensity occurs at ∼6.5 min. The initial burst in intracellular calcium level is then followed by a recovery phase were calcium levels return to near basal levels after 15 min to 35 min for most cells (Fig. 3I andJ), as generally observed for receptor coupled to the calcium pathway. In contrast, the MCWG micrographs (Fig. 3D andE) show a distinctly heterogeneous behavior within the cells with both localized increases and decreases in signal levels, indicating a re-modeling of intracellular structures associated with cell activation 14 and possibly as a result of AT1 receptor-signaling activity. 21,40 Version of the laboratory Please find the article on Analyst website -https://doi.org/10.1039/C8AN00911B Version of the laboratory Please find the article on Analyst website -https://doi.org/10.1039/C8AN00911B Fig. 3 Simultaneous monitoring of intracellular calcium by SEF imaging and cellular morphological response by MCWG imaging (A) reflectance micrograph of 8 individual HEK-AT1 cells; (B-E) reflectance variation micrographs showing local changes in reflectance following stimulation with 100 nM AngII at t = 0 min (blue end of the spectrum indicates a decrease, red end of the spectrum indicates an increase, green indicates no change); (F-J) simultaneous SEF micrographs, where cells were loaded with calcium indicator Fluo-8; (K) plots of the full-time sequence of average MCWG reflected intensity and SEF average intensity, normalized by cell surface area.

The plots in Fig. 3K show the time sequences of averaged MCWG reflected intensity and SEF average intensity, with both signals normalized by cell surface area as presented in Fig. 3A andF. Note that some cells show a certain degree of signal variation during the baseline phase as well as during the onset of the media exchange. Such drifts could be associated with minute changes in the fluid cell temperature or with flow induced effects from the media exchange. The plots show a biphasic response in both the SEF and the MCWG time profiles. Phase I is associated with the initial burst of intracellular calcium following stimulation of the cells with AngII (5 min), confirming the receptor activation and subsequent Gq signaling known to mobilize calcium from the endoplasmic reticulum. 28 Phase II of the reflectance signal is associated in time to the recovery of the intracellular calcium levels for all cells. Intriguingly, these oscillations in calcium levels were not found to be associated to the MCWG signal, indicating that they do not induce significant morphological changes or cell body contraction in this cell model. It should be noted here that oscillating calcium signals are a well-known phenomenon and provide a digital signal to downstream effectors, involved in the regulation of gene expression, a process that normally occurs over several hours. 41 As expected, the SEF and MCWG data are strongly correlated, confirming the receptor-dependent cell signaling response origin of the MCWG label-free signal. However, significant differences can also be observed when comparing individual responses. During phase I, though all cells showed an intracellular calcium burst (indication of AT1R functionality), only cells #1-4 and 7 showed a subsequent marked decrease in average MCWG reflectance. During phase II, cells #1, #5, #7 and #8 exhibited oscillations in their intracellular calcium levels with varying periodicity (116 s, 230 s, 265 s and 173 s, respectively), while cells #2, #3 and #4 showed sustained elevated calcium levels. The absence of such distinct features within the MCWG signal further demonstrates that changes in the intracellular calcium levels seem to not interfere with the observed MCWG signal, which has also been demonstrated for other label-free detection modalities. 21 These observations indicate that the Gq-dependent calcium signal alone is be insufficient to explain the first phase of the MCWG signal, indicating that other pathways may be required for the phase I of the MCWG signal.

Role of ROCK and G12/13 during AT1 signaling and link to cellular activity

In addition to the calcium-dependent Gq signaling cascade, AT1R is also coupled to a calciumindependent G12/13 signaling pathway. 39 The activation of this pathway ultimately leads to activation of ROCK, which is a ubiquitous intracellular kinase 38 involved in actin remodeling and the regulation of actomyosin contractile activity via myosin light chain (MLC) phosphorylation and inhibition of the myosin light chain phosphatase (MLCP). To test the involvement of G12/13 Version of the laboratory Please find the article on Analyst website -https://doi.org/10.1039/C8AN00911B signaling and thus the involvement of ROCK in the measured cellular response, HEK293-AT1R cells were treated with the ROCK inhibitor Y-27632 prior to activation with AngII. Fig. 4A-J show MCWG (A: reference, B-E: differences) and SEF micrographs from the experiment, alongside plots of cell-specific average MCWG and SEF responses for the time series normalized by cell area (Fig. 4K). As expected, the MCWG data do not show a signal decrease (phase I) following AT1 activation, confirming the robust suppression of cell signaling activity through ROCK inhibition (Fig. 4C). The phase II in the MCWG signal response nevertheless appears not to be impacted as indicated by the appearance of large yellow and red areas in the MCWG micrographs (Fig. 4D andE). The SEF data again shows a robust release of intracellular calcium, as expected from AT1 activation (Fig. 4G-J). When comparing the experimental time series (Fig. 4K), interestingly despite the absence of a Phase I response in the MCWG data, all cells nevertheless show a steady increase in average MCWG response during Phase II suggesting a distinct G12/13independent signaling mechanism for Phase II compared to the previous experiment due to additional unidentified component. The SEF time series shows again a biphasic response profile where all cells respond with a transient release of intracellular calcium shortly after AngII stimulation as expected from AT1 activation. Overall, these results indicate that G12/13 signaling may be the major signaling pathway responsible for Phase I and is consistent with previous population-based findings using SPR spectroscopy in which ROCK inhibition, but not MLCK inhibition, associated with Gq signaling could successfully suppress Phase I contractile response.

Conclusion

In this work, we demonstrated a combined MCWG and SEF microscopy system for dual mode imaging and signal analysis of receptor-dependent cell signaling activity. We demonstrated the capabilities of this system in experiments designed to (1) identify the specific structural components of the cytoskeleton imaged by MCWG and SEF, and (2) provide insight into intracellular signaling following GPCR activation in living cells. In the latter case, SEF imaging revealed the intracellular Gq-dependent calcium mobilization while simultaneous MCWG imaging showed the distinct individual cell response profiles to AngII-mediated AT1R activation. A strong temporal correlation was observed between the MCWG label-free signal and the labelled calcium signal from individual cells, confirming the origin of the MCWG label-free signal as a receptordependent cell signaling response. Interestingly, a discrepancy between the two signals observed for some cells suggest that the cell signaling activity measured through the MCWG signal channel is not necessarily dependent on calcium mobilization. This type of observation is only possible through a comparative analysis of individual cell heterogeneity present in the two distinct response profiles. Using a selective G12/13/Rho/ROCK signaling inhibitor, we determined that this pathway is required for the phase I response observed with MCWG imaging. Interestingly, the second phase of the response profile was not impacted by G12/13/Rho/ROCK signaling inhibition, suggesting that other pathways such as Gq-dependent pathway may be required for this later phase. While other signaling cascades could also be involved in AT1 signaling, this work shows how individual cell assessment using MCWG and SEF together with pharmacological tools and fluorescent probes targeting specific cell signaling pathways can enables the identification of key contributors to a given cellular response. Thus, this system should find applications in drug screening, signaling cascade profiling, and in the study of fundamental cellular mechanisms.

13

 13

Fig. 1

 1 Fig. 1 Combined MCWG and SEF imaging platform. MCWG imaging is based on a highnumerical-aperture objective. SEF fluorescence emission is collected from the top by a long working distance objective.

Fig. 2

 2 Fig. 2 Vascular smooth muscle cell simultaneously imaged by MCWG and SEF. (A) MCWG micrographs of a single VSMC taken at different angles of incidence. The black square in the leftmost image represents the location of ROI used to calculate average reflected intensity, while the black arrow indicates the direction of mode propagation; (B) SEF micrographs simultaneously taken at the same angles of incidence, F-actin was labeled with Alexa Fluor 532 Phalloidin; (C) plots of normalized average reflected light intensity within the ROIs in A and overall average SEF intensity in B as a function of incidence angle.

21

 21 Fig.4A-Jshow MCWG (A: reference, B-E: differences) and SEF micrographs from the experiment, alongside plots of cell-specific average MCWG and SEF responses for the time series normalized by cell area (Fig.4K). As expected, the MCWG data do not show a signal decrease (phase I) following AT1 activation, confirming the robust suppression of cell signaling activity through ROCK inhibition (Fig.4C). The phase II in the MCWG signal response nevertheless appears not to be impacted as indicated by the appearance of large yellow and red areas in the MCWG micrographs (Fig.4D and E). The SEF data again shows a robust release of intracellular calcium, as expected from AT1 activation (Fig.4G-J). When comparing the experimental time series (Fig.4K), interestingly despite the absence of a Phase I response in the MCWG data, all cells nevertheless show a steady increase in average MCWG response during Phase II suggesting a distinct G12/13independent signaling mechanism for Phase II compared to the previous experiment due to additional unidentified component. The SEF time series shows again a biphasic response profile where all cells respond with a transient release of intracellular calcium shortly after AngII stimulation as expected from AT1 activation. Overall, these results indicate that G12/13 signaling may be the major signaling pathway responsible for Phase I and is consistent with previous population-based findings using SPR spectroscopy in which ROCK inhibition, but not MLCK inhibition, associated with Gq signaling could successfully suppress Phase I contractile response.21

Fig. 4

 4 Fig. 4 Impact on ROCK inhibition during AT1 analyzed by MCWG and SEF microscopy. (A) Reflectance micrograph of individual HEK-AT1R cells; (B-E) reflectance variation micrographs showing local changes in reflectance after pretreatment with Y-27632 and stimulation with 100 nM AngII; (F-J) SEF micrographs of the same cells, loaded with calcium indicator Fluo-8; (K) plots of cell-specific average MCWG and SEF responses for the full time series normalized by cell area.

Acknowledgements

This work was supported by the Fonds de recherche du Québec -Nature et technologies (FRQNT: MG, PGC, TS), the Natural sciences and engineering research council of Canada (NSERC: MG, PGC, MC), and the Canadian Institutes of Health Research (CIHR) under the Collaborative Health Research Program (MG, PGC). LN2 is an international research laboratory co-managed in Canada by University of Sherbrooke and in France by CNRS, Université de Lyon (Centrale Lyon, INSA Lyon et CPE Lyon), and Université Grenoble-Alpes; LN2 is funded in Québec by the FRQNT. TS was also supported by the COMET K1 center Austrian Smart Systems Integration Research (ASSIC). The Version of the laboratory Please find the article on Analyst website -https://doi.org/10.1039/C8AN00911B COMET Competence Centers for Excellent Technologies Program is supported by BMVIT, BMWFW and the federal provinces of Carinthia and Styria of Austria.

Conflicts of interest

There are no conflicts to declare.