Thèse présentée pour obtenir le grade de Docteur de l'Université Louis Pasteur

Patrick Pirrotte

Description

Niveau: Supérieur, Doctorat, Bac+8
.. . . . . . . . . Thèse présentée pour obtenir le grade de Docteur de l'Université Louis Pasteur Strasbourg I Discipline : Science du Vivant par Patrick Pirrotte Applications biologiques du NanoSIMS Soutenue publiquement le 27 novembre 2007 Membres du jury Directeur de Thèse : M. François Lasbennes, Professeur, Université Louis Pasteur Rapporteur Interne : M. Jean-Pierre Bucher, Professeur, Université Louis Pasteur Rapporteur Externe : M. Ron M. Heeren, Professeur, Université d'Utrecht Rapporteur Externe : M. Alain Croisy, Docteur, Institut Curie Directeur de Thèse : M. Claude P. Muller, Professeur, Université de Trèves

• fond national de la recherche


• sims


• immunology who


• centre de recherche public


• ministère de la culture, de la recherche et de l'enseignement supérieur


• secondary ion


• rio d'imagerie cellulaire

Sujets

• Louis Pasteur University
• Bûcher
• Turner
• Bailly
• Institut Curie
• Lippmann
• Kremer

Informations

• Publié par: profil-zyak-2012
• Publié le: 01 novembre 2007
• Nombre de lectures: 56
• Langue: English
• Poids de l'ouvrage: 22 Mo

Extrait

. . . . . . .

Thèse présentée pour obtenir le grade de Docteur de l’Université Louis Pasteur Strasbourg I

Discipline : Science du Vivant par Patrick Pirrotte .

Applications biologiques du NanoSIMS

Soutenue publiquement le 27 novembre 2007

Membresdu jury

.

.

Directeur de Thèse : M. François Lasbennes, Professeur, Université Louis Pasteur Directeur de Thèse : M. Claude P. Muller, Professeur, Université de Trèves Rapporteur Interne : M. Jean-Pierre Bucher, Professeur, Université Louis Pasteur Rapporteur Externe : M. Ron M. Heeren, Professeur, Université d’Utrecht Rapporteur Externe : M. Alain Croisy, Docteur, Institut Curie

This doctoral thesis has been performed at the Département d’Immunologie

Laboratoire National de Santé, Luxembourg

under the guidance of

Professor Claude P. Muller, Département d’Immunologie, Laboratoire National de Santé,

Luxembourg

and

Professor François Lasbennes, Institut des Neurosciences Cellulaires et Intégratives,

Strasbourg

Part of this work was performed at

Centre de Recherche Public – Gabriel Lippmann – Science and Analysis of Materials,

Luxembourg

Centre de Recherche Public – Santé, Luxembourg

Plateau technologiquede l’IFR 37 des Neurosciences, Strasbourg

Plateforme RIO d’Imagerie Cellulaire Strasbourg Esplanade

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Acknowledgments

I am deeply indebted to my promoter Professor Claude P. Muller who allowed me to join his team at theDepartment of Immunology of theLaboratoire National de Santé, Luxembourg. He managed to provide excellent scientific guidance and support whilst allowing me to develop my own ideas. I thank my second promoter Professor François Lasbennes from theInstitut des Neurosciences Cellulaires et Intégratives, Strasbourg for his interest in the project and the unyielding support he gave me in the last 6 years to further my career in Biological Imaging. I would like to express my gratitude towards the staff of theCentre de Recherche Public - Gabriel Lippmann Science and Analysis of Materials,Luxembourg,Dr Jean-Nicolas Audinot, operator of the NanoSIMS, Dr Henri-Noël Migeon, Jérôme Demange and Esther Lentzen for sharing their expertise and for allowing me to use the facilities available in their laboratory. Most of the electron microscopy work presented in this thesis was performed at theplateau technologiquede l’IFR 37 des Neurosciences, Strasbourg.I am grateful to Valérie Demaïs and Dr Yannick Bailly for their assistance in developing this technique. I would also like to thank Dr Matthieu Erhardt from theplateforme RIO d’Imagerie Cellulaire Strasbourg Esplanadefor his help in analysing lichen samples by electron microscopy. I would like to thank Dr Emmanuel Prodhomme and Dominique Revets for their scientific support and encouragement, their critical reading of the manuscript and their continuous help in fighting daily frustrations. I would also like to thank Sandrina Azevedo for introducing me to the interesting world of lichens and Ana Fernández-Salegui for providing samples from her lichen study for me to analyze on NanoSIMS. Special thanks go to Dr Jérôme Mutterer without whom I probably would not have discovered the joys of digital image processing and whose knowledge on confocal microscopy enlightened me. ii

I am indebted to Professor André Steinmetz and Dr Céline Hoffmann for granting me access to the confocal platform of theCentre de Recherche Public – Santé, Luxembourgand for their useful advices. A special mention goes to Dr Jean-Luc Guerquin-Kern from theCentre de Recherche Imagerie intégrative : de la molécule à l'organismethe at Institut Curie, Parishis for insightful ideas in SIMS. I acknowledge theCentre de Recherche Public – Santé, theFond National de la Recherche, Ministère de la Culture, de la Recherche et de l’Enseignement Supérieurthe and Fondation Reinert-Schwachtgentrusted me with a research scholarship. Without their financial that assistance, this research would not have been possible. Many thanks go to Claude Schwachtgen for the proofreading of the manuscript and to Daniel Toth for his help in printing this manuscript. I would also like to acknowledge my colleagues from theDepartment of Immunology who shared their knowledge and expertise with me, amongst them most specially Wim Amerlaam, Dr Jacques Kremer, Dr Stefan de Buck, Sophie Farinelle, Stéphanie Willième, Julia Kessler, Dr Fabienne Bouche, Dr Jonathan Turner. Special thanks go to Dr Fred Fack, whose encyclopedic knowledge was a fountain of challenging ideas. I am grateful to Ulla Muller and Carole Weis for their continuous assistance. I would like to thank Joana for all her constant support, encouragement and so much more. I am deeply indebted to my parents, who supported me patiently for all these years.

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Abstract

Secondary Ion Mass Spectrometry (SIMS) is based upon the sputtering of a few atomic layers from the surface of a sample, induced by a ’primary ion’ bombardment. An energetic primary ion impact triggers a cascade of atomic collisions resulting in an erosion of atoms and molecules. Some of the ejected particles can be spontaneously ionized and are representative of the target area composition. In a SIMS instrument, these “secondary ions” are accelerated and separated in function of their mass/charge ratio (m/z) before detection. The NanoSIMS is a dynamic SIMS ion microprobe capable of imaging the distribution of elemental ions at high lateral resolution (50 nm) with a high mass resolution. The aim of this thesis was to define the requirements of such instruments, to evaluate their utility in life sciences and to develop potential applications in biology. In order to do so, preparative and analytical methods had to be devised to improve ion imaging by SIMS. The limited knowledge about the behaviour of biological samples under a primary ion beam impeded the identification of tissue and cellular features and required supplementary confirmation by complementary techniques. The ability of SIMS to discriminate isotopes of a same element has encouraged us to develop isotopically labelled biomolecules of varying specificity. Abiding to the experience gained in the course of the thesis, several applications are proposed in the areas of trace metal detection, antigen uptake and protein localization. Finally, NanoSIMS is compared to other techniques of microanalytical imaging and the role of the ion microprobe in life sciences is discussed as well as its prospects. Keywords: SIMS, NanoSIMS, imaging, stable isotope, 15-Nitrogen, immunolabelling, pulse chase, metabolic labelling, trace metal detection, implantation, ratiometric analysis

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List of abbreviations

AA ABC APC ATCC Beta BrdU BSA

CHAPS CLSM DHB DMSO dSIMS EDTA FBS Fitc GAPDH HA HCCA LNS MALDI MOI MV MW NP NS50 OM PBS PI PMA PMF PSD SAM SEM SIMS sSIMS TEM TFA TOF

Amino acid Ammonium bicarbonate buffer Antigen presenting cells American Type Culture Collection Beta-actine 5’-bromo-2-deoxyuridine Bovine serum albumine 3-[(3-Cholamidopropyl)dimethylammonio]-1- propanesulfonate Confocal Laser Scanning Microsope 2,5-dihydroxybenzoic acid Dimethyl Sulfoxide Dynamic secondary ion mass spectrometry Ethylenediaminetetraacetic acid Fetal bovine serum Fluorescein isothiocyanate Glyceraldehyde-3-phosphate dehydrogenase Hemagglutinin α-Cyanohydroxycinnamic acid Laboratoire National de Santé Matrix assisted Laser Desorption Ionization Multiplicity of infection Measles Virus Molecular weight Nucleoprotein Cameca NanoSIMS 50 Optical microscope Phosphate buffered Saline Propidium iodide Phorbol myristate acetate Protein mass fingerprint Post-source decay Service Analyse des Matériaux Scanning Electron Microscope Secondary ion mass spectrometry Static secondary ion mass spectrometry Transmission Electron Microscope Trifluoroacetic acid Time of flight

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Table of Contents

Acknowledg................................i..i............s..ment................................................

Abstract ............................................................................................................... iv

List of abbreviations............................................................................................ v

Figure Index ........................................................................................................ ix Table Index ......................................................................................................... xi Résumé de thèse de doctorat ............................................................................xii Chapter IIntroduction..................................................................................... 1 I.A.Secondary Ion Mass Spectrometry 4 I.A.1. General SIMS Principle ..................................................................................... 4 I.A.2. Sputtering, Ionization, Implantation................................................................... 5 I.A.3. Secondary ion intensity and ionization efficiency ............................................. 7 I.A.4. Primary ion sources ............................................................................................ 9 I.A.5. The acceleration of secondary ions and their separation according to the ratio mass/charge ...................................................................................................................... 10 I.A.6. The detection of secondary ions....................................................................... 11 I.B.The NanoSIMS 50 12 I.B.1. Geographical distribution, price and use of the NanoSIMS............................. 18 I.B.2. Sample preparation........................................................................................... 20 I.C.28Review of current biological applications in dSIMS and NanoSIMS I.C.1. Cell biology ...................................................................................................... 28 I.C.2. Pharmacology................................................................................................... 29 I.C.3. Toxicology ....................................................................................................... 31 I.C.4. Biomineralization ............................................................................................. 32 I.C.5. Radiotoxicology and nuclear medicine ............................................................ 33 I.D.Imaging techniques directly related to SIMS 34 I.D.1. TOF-SIMS imaging.......................................................................................... 34 I.D.2. MALDI-TOF imaging...................................................................................... 35 I.E.Confocal Microscopy 35 I.E.1. Introduction ...................................................................................................... 35 I.E.2. Principles and Instrumentation......................................................................... 36 I.E.3. Resolution in optical microscopy ..................................................................... 38 I.E.4. Fluorescent probes............................................................................................ 39 I.E.5. General sample preparation considerations...................................................... 40 I.E.6. Advantages and disadvantages of confocal microscopy .................................. 41 I.F.Electron microscopy 42 I.F.1. Introduction ...................................................................................................... 42 I.F.2. Principles and Instrumentation......................................................................... 43 I.F.3. Instruments related to TEM.............................................................................. 44 I.F.4. Resolution in electron microscopy ................................................................... 45 I.F.5. General sample preparation considerations...................................................... 46 I.G.46Matrix Assisted Laser Desorption Ionization – Time of flight (MALDI-TOF) I.G.1. Introduction ...................................................................................................... 46 I.G.2. Principle and Instrumentation .......................................................................... 47 I.G.3. General sample preparation considerations...................................................... 49 vi

I.H.49Scope of the thesis Chapter IISoftware Development .............................................................. 52 II.A.Development of an image analysis software package for Cameca NanoSIMS images 53 II.A.1. Introduction ...................................................................................................... 53 II.A.2. Developmental notes ........................................................................................ 54 II.B.Enhancement of SIMS images 59 II.C.Development of an image analysis software package for Zeiss LSM510 confocal images 62 II.D.Discussion 64 Chapter IIIImplantation on NanoSIMS ..................................................... 66 III.A.Introduction 67 III.B.Material and Methods 69 + III.B.1. Computer simulation of Cs implantation........................................................ 69 III.B.2. Sample preparation........................................................................................... 70 III.B.3. Analytical conditions........................................................................................ 71 III.B.4. Preimplantation ................................................................................................ 72 III.C.Results 73 + III.C.1. Computer simulation of Cs implantation........................................................ 73 III.C.2. Dose variation and its effect on imaging.......................................................... 77 III.C.3. The dynamic regime as a function of the implanted dose: Relationship to the surface 79 III.C.4. Variation of implantation primary ion beam current ....................................... 83 III.C.5. Secondary ion emission after pre-implantation on the IMS-6f ........................ 83 III.D.Discussion 85 Chapter IVInvestigation of general imaging procedures on NanoSIMS 88 IV.A.Introduction 89 IV.B.Materials and Methods 89 IV.B.1. Sample preparation of murine lung, kidney and peyer patches for ion microprobe analysis.......................................................................................................... 89 IV.B.2. Sample preparation of rat brain and spinal cord for ion microprobe analysis . 90 IV.B.3. Sample preparation for nuclear labelling of metaphase arrested cells using BrdU 91 IV.C.Results 91 IV.C.1. Ion microprobe analysis of Peyer patches........................................................ 92 IV.C.2. Ion microprobe analysis of murine lung .......................................................... 93 IV.C.3. Ion microprobe analysis of murine kidney....................................................... 94 IV.C.4. Ion microprobe analysis of rat cerebellum and spinal cord ............................. 95 IV.C.5. Ion microprobe analysis of BrdU labelled metaphase arrested cells ............... 98 IV.D.Discussion 100 Chapter VNanoSIMS imaging of trace metals in lichens...................... 101 V.A.Introduction 102 V.B.103Materials and Methods V.C.Results 104 V.D.Discussion 108 Chapter VIFollowingthephagocytosis of a metabolicallylabelled antigen by NanoSIMS ................................................................................................... 110 VI.A.Introduction 111 vii

VI.B.Materials and Methods: 111 15 VI.B.1. N / BrdU labelling of E. coli........................................................................ 111 VI.B.2. Fitc fluorescent labelling of E. coli ................................................................ 112 15 VI.B.3. Assessment of the N labelling efficiency of E. coli by MALDI-TOF mass spectrometry ................................................................................................................... 113 VI.B.4. Assessment of the THP-1 phagocyte function by flow cytometry................. 114 15 VI.B.5. Assessment of the N labelling efficiency of E. coli by NanoSIMS. Imaging of THP-1 phagocytic activity. ............................................................................................ 115 VI.C.Results 116 VI.C.1. Assessment of the phagocyte function of THP-1 by flow cytometry ............ 116 15 VI.C.2. Assessment of the N labelling efficiency of E. coli by MALDI-TOF mass spectrometry ................................................................................................................... 119 15 VI.C.3. Assessment of the N labelling efficiency of E. coli by NanoSIMS. Imaging of THP-1 phagocytic activity. ............................................................................................ 123 VI.D.Discussion 129

Chapter VIIMetabolicpulse labellingand immunolabellingin a viral study using NanoSIMS.................................................................................... 131 VII.A.Introduction 132 VII.B.Materials and Methods 132 VII.B.1. Timeline of a MV infection........................................................................ 132 VII.B.2. Immunofluorescence assay ........................................................................ 134 VII.B.3. Stable isotope pulse labelling of MV infected THP-1 cells ....................... 136 VII.B.4. Stable isotope labelling of an antibody directed against a viral protein..... 136 VII.B.5. Immunolabelling assay by NanoSIMS....................................................... 138 VII.C.Results 139 VII.C.1. Timeline of a MV infection........................................................................ 139 VII.C.2. Immunofluorescence assay ........................................................................ 143 VII.C.3. Stable isotope pulse labelling of MV infected THP-1 cells. ...................... 145 VII.C.4. Stable isotope labelling of an antibody directed against a viral protein..... 148 VII.C.5. Immunolabelling assay by NanoSIMS....................................................... 153 VII.D.Discussion 154

Chapter VIII

General Discussion............................................................... 155

References ........................................................................................................ 165

Annexe .................................................................................................................. a Résumé de thèse de doctorat (court) b Secondary Ion Mass Spectrometry in life sciences g Résumé x

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Figure Index

Figure 1: Imaging methods compared by their timescales, penetration depths and ranges of lateral dimensions....................................................................................................................... 3 Figure 2: Primary ion beam particles (atoms or clusters) impacting on the sample surface...... 6 Figure 3: Schematic representation of the conventional probe forming configuration............ 10 Figure 4: The double focusing mass analyzer, a combination of an electrostatic sector and a magnetic sector......................................................................................................................... 11 Figure 5: The NanoSIMS 50 at the Centre de Recherche Public Gabriel Lippmann in Luxembourg. ............................................................................................................................ 12 Figure 6: Schematic representation of a co-axial probe forming configuration ...................... 14 Figure 7: The Cameca Microbeam caesium source ................................................................. 14 Figure 8: The duoplasmatron source ........................................................................................ 15 Figure 9: Schematic representation of the NanoSIMS............................................................. 16 Figure 10: Schematic representation of a confocal laser scanning microscope ....................... 37 Figure 11: One-step immunolabelling procedure..................................................................... 41 Figure 12: Two-step immunolabelling procedure. ................................................................... 41 Figure 13: Schematic representation of a TEM ....................................................................... 44 Figure 14: Principle of PSD and reflectron .............................................................................. 48 Figure 15: Susceptible peptide backbone cleavage sites.......................................................... 48 Figure 16: Representation of the Hue, Saturation and Intensity colour gradient. .................... 57 Figure 17: SIMSToolbox interface in ImageJ. Ratio graphical user interface and info screen. .................................................................................................................................................. 58 Figure 18: SIMSToolbox Metadata tree representation, with the XML export option............ 58 Figure 19: Inhomogeneously implanted Ion micrographs and their histograms. Enhancement by histogram matching algorithms. .......................................................................................... 61 Figure 20: LSMToolbox interface in ImageJ, with info screen, metadata tree representation and the Lut_Panel gradient window......................................................................................... 63 Figure 21: Inhomogeneously implanted images and their intensity profiles. .......................... 69 Figure 22: Vacuum transfer suitcase. ....................................................................................... 72 Figure 23: SRIM modelling principle. ..................................................................................... 73 Figure 24: TRIDYN modelling principle................................................................................. 74 Figure 25: Erosion versus Dose modelled using TRIDYN...................................................... 75 2 Figure 26: Concentration (in atomic fractions) versus Dose (in atoms/cm ) modelled using TRIDYN................................................................................................................................... 76 Figure 27: Secondary emission Yield (ratio of secondary ions emitted over incident Cs+ 2 primary ions) versus Dose (in atoms/cm ) using TRIDYN. .................................................... 77 Figure 28: Ion maps of THP-1 cells (20 µm x 20 µm) after varying implantation times. ....... 78 Figure 29: Depth profile (30 µm x 30 µm, 160 pA ) of a THP-1 cell suspension embedded in epoxy resin. .............................................................................................................................. 79 Figure 30: Depth profiles of a selection of biologically relevant ions at increasing surfaces, in cells and kidneys. ..................................................................................................................... 81 Figure 31: Time to reach the equilibrium, plotted versus the implantation surface................. 82 Figure 32: Ion micrographs of cells after 2 nA implantation. .................................................. 83 Figure 33: Depth profile analysis of pre-implanted resin embedded mouse kidney on NanoSIMS. ............................................................................................................................... 84 Figure 34: Kidney sample, implantation on the IMS-6f, vacuum suitcase transfer to the NanoSIMS. ............................................................................................................................... 85 Figure 35: Ion micrograph of murine Peyer patches................................................................ 93

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