The novel approach was proposed for detailed high-resolution studies of morphology and physico-chemical properties concomitantly at one measurement spot of E. coli bacterial cells culture immobilized onto silicon wafer surface in UHV conditions applying PhotoEmission Electron Microscopy under Hg lamp irradiation. For the E. coli characterization scanning electron microscopy (electron beam) and X-ray photoelectron spectroscopy (X-ray tube radiation) were applied prior to PhotoEmission Electron Microscopy measurements. In spite of irradiation doses collected for the cell arrays we were successful in detection of high-resolution images even of single E. coli bacterium by PhotoEmission Electron Microscopy technique followed by detailed high-resolution morphology studies by scanning electron microscopy. These results revealed widespread stability of the E. coli membranes shape after the significant number of applied characterization techniques. © 2019 The Author

Antipov, S.S.

Belikov, E.A.

Berezhnoy, A.A.

Chumakov, R.G.

Chuvenkova, O.A.

Koyuda, D.A.

Kulikova, T.V.

Lebedev, A.M.

Marchenko, D.

Parinova, E.V.

Praslova, N.V.

Preobrazhenskaya, E.V.

Sivakov, V.

Turishchev, S.Yu.

Valiakhmedova, I.V.

Repositorium für Naturwissenschaften und Technik  (TIB Hannover)

Contents lists available at ScienceDirectResults in Physicsjournal homepage: www.elsevier.com/locate/rinpMicroarticleOn the possibility of PhotoEmission Electron Microscopy for E. coli advancedstudiesS.Yu. Turishcheva,⁎, D. Marchenkob, V. Sivakovc, E.A. Belikova, O.A. Chuvenkovaa,E.V. Parinovaa, D.A. Koyudaa, R.G. Chumakovd, A.M. Lebedevd, T.V. Kulikovaa, A.A. Berezhnoya,I.V. Valiakhmedovaa, N.V. Praslovaa, E.V. Preobrazhenskayae, S.S. Antipova,e,fa Voronezh State University, 394018, Universitetskaya pl.1, Voronezh, RussiabHelmholtz-Zentrum Berlin, 12489, Albert-Einstein-Str. 15, Berlin, Germanyc Leibniz Institute of Photonic Technology, 07745, Albert-Einstein-Str. 9, Jena, GermanydNational Research Center “Kurchatov Institute”, 123182, Akademika Kurchatova pl. 1, Moscow, Russiae Institute of Cell Biophysics RAS, 142290, Institutskaya St. 3, Pushchino, Moskovskaya obl., Russiaf Immanuel Kant Baltic Federal University, 236016, A. Nevskogo St. 14, Kaliningrad, RussiaA R T I C L E I N F OKeywords:SpectromicroscopyPhotoemission electron microscopyScanning electron microscopyX-ray photoelectron spectroscopySurface compositionBioimagingE. coliA B S T R A C TThe novel approach was proposed for detailed high-resolution studies of morphology and physico-chemicalproperties concomitantly at one measurement spot of E. coli bacterial cells culture immobilized onto siliconwafer surface in UHV conditions applying PhotoEmission Electron Microscopy under Hg lamp irradiation. Forthe E. coli characterization scanning electron microscopy (electron beam) and X-ray photoelectron spectroscopy(X-ray tube radiation) were applied prior to PhotoEmission Electron Microscopy measurements. In spite of ir-radiation doses collected for the cell arrays we were successful in detection of high-resolution images even ofsingle E. coli bacterium by PhotoEmission Electron Microscopy technique followed by detailed high-resolutionmorphology studies by scanning electron microscopy. These results revealed widespread stability of the E. colimembranes shape after the significant number of applied characterization techniques.The combination of a few nanometers inorganic particles withnature-like objects as a hybrid nanostructure can be a breakthroughway for the modern applications developing driven by novel low-costand energy effective technology approaches of functional low-dimen-sional materials formation [1–5]. One of the convenient objects for suchkind of technology development is E. coli bacterial cells that can beeasily grown in simple and controlled laboratory conditions [6–8].Knowledge of surface properties and composition plays an importantrole for such systems so the precision control is necessary to be applied.Scanning Electron Microscopy (SEM) is one of the most developed anduseful tool for morphology studies of different functional materials[9–11] including biosystems such as based on E. coli arrays [12–15].Nevertheless, a precise and chemically sensitive microspot surfacestudies on biological objects are still not well investigated field. Pho-toEmission Electron Microscopy (PEEM) in coupling with laboratory orsynchrotron based excitation sources can play a keyrole for such tasks[16–18]. PEEM technique provides the unique and powerful ability toperform chemically sensitive (selective) microspot spectroscopy andmicroscopy in one experiment run [19–21]. The crucial point for PEEMstudies of bio-objects is their stability under special conditions of sur-face sensitive experiments.E. coli K12 MG1665 bacteria colony were grown aerobically in LBmedium at 37 °C with constant mixing up to the stationary growthphase [7] and precipitated by centrifugation (2500 rpm) then washedtwice with TE-NaCl buffer solution (50 mM Tris-HCl pH 7.5; 0.1 mMEDTA; 50 mM NaCl) and twice with distilled water. The washed cellswere deposited to a cleaned silicon surface (20, 40, or 60 μl), dried at37 °C and washed three times with distilled water. For preliminary SEMstudies JEOL JSM-6380LV microscope was used while for high resolu-tion images Carl Zeiss ULTRA 55 microscope was applied. UHV X-rayphotoelectron spectrometer was equipped with high resolution SPECSPhoibos 150 hemispherical electron energy analyzer with monochro-matic X-ray source. Energy resolution was found in the range of 0.1 eVwith the analysis depth estimation of about 3 nm. The calibration of thespectra was carried out using a signal of pure gold film and the positionof Au 4f level. Omicron Focus PEEM microscope of Russian-German Labat Helmholtz-Zentrum Berlin was used for PEEM studies.Herein for the first time we present PEEM results for E. coli K12https://doi.org/10.1016/j.rinp.2019.102821Received 27 October 2019; Received in revised form 14 November 2019; Accepted 17 November 2019⁎ Corresponding author.E-mail address: tsu@phys.vsu.ru (S.Y. Turishchev).Results in Physics 16 (2020) 102821Available online 22 November 20192211-3797/ © 2019 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/BY-NC-ND/4.0/).TMG1665 cell line in combination with X-ray photoelectron spectro-scopy (XPS) control that was preceded and followed by SEM studies.SEM studies were performed to control the cells membrane morpho-logical stability after accumulated electrons and photons radiationdoses during the PEEM and XPS studies. For all consistently performedmeasurements the initial probes were stored for more than two monthsin the ambient condition being deposited on clean silicon surfaces XPSstudies were performed under Al Kα radiation (1486.61 eV), SEMmeasurements at accelerating voltages of 2, 5 and 20 kV. Aiming atreducing the radiation damage of cells array during the PEEM mea-surements we used Hg lamp with excitation energy of ~5 eV.Initial SEM images are revealed distribution of E. coli bacteria lo-calized on the silicon surface with a well-known shape and othercharacteristics (e.g. sizes) of a single bacterium [12–14]. XPS verifica-tion spectrum (Fig. 1) confirms the absence of any noticeable surfacecontamination. All elements peculiar to the deposited bacterial cellsarrays are observed such as carbon, nitrogen and oxygen together withbuffer and working solutions residuals (Na, Cl, etc). Finally, Si corelevels are detected from the substrate because of silicon substratesfragmentary coverage by E. coli array. All observed lines were identifiedat XPS spectrum given in the Fig. 1.PEEM images (Fig. 2) demonstrate a possibility of effective bioi-maging of the E. coli bacterial cells array using such type of visualiza-tion technique. Moreover, well distinguished observation of a singlebacterium is detected by PEEM technique that correlates well with SEMresults. The quality of the E. coli bacterial cells surface morphologyfeatures are nearly the same as compared with SEM image (Fig. 2b).SEM studies performed after XPS (X-ray irradiation) and PEEM (Hglamp irradiation) experiments demonstrate only partial bacteria mem-brane destruction. Only partial destruction of the bacteria membraneconfirms the surprisingly strong stability of E. coli MG1665 culture andits fragments. These easily observed dark parts of a single observed cellsmembrane can be detected in SEM and even more in PEEM images.These results suggest promising perspective of nanobiofunctional ma-terials effective synchrotron studies [20–23] under feasibly low-dosephotons excitation for microspot chemically sensitive PEEM (i.e. spec-tromicroscopy) surface analysis possibly performed in UHV conditionthat as shown in present paper can be applied up to a single E. colibacterial cell bioimaging.CRediT authorship contribution statementS.Yu. Turishchev: Conceptualization, Methodology, Investigation,Writing - original draft, Writing - review & editing, Supervision, Projectadministration, Funding acquisition. D. Marchenko: Methodology,Validation, Investigation, Writing - review & editing. V. Sivakov:Methodology, Validation, Investigation, Writing - review & editing,Funding acquisition. E.A. Belikov: Validation, Formal analysis,Resources. O.A. Chuvenkova: Methodology, Formal analysis,Investigation, Visualization. E.V. Parinova: Validation, Formal ana-lysis, Investigation. D.A. Koyuda: Validation, Formal analysis,Investigation. R.G. Chumakov: Methodology, Validation,Investigation, Writing - review & editing. A.M. Lebedev: Methodology,Validation, Investigation, Writing - review & editing. T.V. Kulikova:Fig. 1. XPS survey spectrum of the E. coli array localized on the Si wafer. Main core level lines are indicated.Fig. 2. High resolution PEEM images (a, two different surface areas) followed by SEM micrograph (b) of the E. coli array localized on the silicon substrate surface.S.Y. Turishchev, et al. Results in Physics 16 (2020) 1028212Methodology, Investigation. A.A. Berezhnoy: Methodology,Investigation, Resources. I.V. Valiakhmedova: Methodology,Investigation, Resources. N.V. Praslova: Methodology, Visualization.E.V. Preobrazhenskaya: Methodology, Investigation. S.S. Antipov:Methodology, Formal analysis, Investigation, Writing - review &editing, Supervision, Funding acquisition.AcknowledgementsThe study was supported by Russian Science Foundation (Project19-72-20180) in part of high resolution XPS and SEM measurements(S.T., O.C., E.P., D.K., T.K., A.B.) and by Ministry of Education andScience of Russia State Tasks (Pr. 16.8158.2017/8.9) in part of PEEMexperiments data evaluation (S.T. and O.C.). Russian-German Labprovided PEEM Microscope facility supported by BMBF grant No.05K12KE1. V.S. acknowledges BMBF in frame of Baltic Sea Network“NanoPhoto” (Grant No. 01DS14017) and DFG (Grant No. SI1893/18-1) for financial support. S.A. funded by the Russian AcademicExcellence Project at the Immanuel Kant Baltic Federal University. Theresults were partially obtained on the equipment of the collective usecenter of Voronezh State University and Kurchatov SynchrotronRadiation Center.References[1] Hashima Yu, Ishikawa Y, Raifuku I, et al. 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On the possibility of PhotoEmission Electron Microscopy for E. coli advanced studies

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On the possibility of PhotoEmission Electron Microscopy for E. coli advanced studies

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