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Confocal angle resolved linear dichroism microscopy for structural fluorescence imaging PDF

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Confocal angle resolved linear dichroism microscopy for structural fluorescence imaging Xiao Wang To cite this version: Xiao Wang. Confocal angle resolved linear dichroism microscopy for structural fluorescence imaging. Optics / Photonic. Centrale Marseille, 2013. English. ￿NNT: ￿. ￿tel-00871010￿ HAL Id: tel-00871010 https://theses.hal.science/tel-00871010 Submitted on 8 Oct 2013 HAL is a multi-disciplinary open access L’archive ouverte pluridisciplinaire HAL, est archive for the deposit and dissemination of sci- destinée au dépôt et à la diffusion de documents entific research documents, whether they are pub- scientifiques de niveau recherche, publiés ou non, lished or not. The documents may come from émanant des établissements d’enseignement et de teaching and research institutions in France or recherche français ou étrangers, des laboratoires abroad, or from public or private research centers. publics ou privés. Centrale Marseille THÈSE pour obtenir le grade de Docteur en Sciences de Centrale Marseille Discipline : Optique, photonique et traitement d’image Confocal angle resolved linear dichroism microscopy for structural fluorescence imaging soutenue publiquement le 25 Septembre 2013 par Xiao WANG École Doctorale : Physique & Sciences de la Matière Rapporteurs : Prof. Markus Sauer Dr. Gilles Tessier Examinateur : Prof. Colin Sheppard, président de jury Directeurs de thèse : Dr. Sophie Brasselet Dr. Patrick Ferrand Institut Fresnel, Aix Marseille Université, CNRS UMR 7249, Centrale Marseille Abstract Based on the fact that the absorption of light is a molecular-orientation sensitive process, fluorescence microscopy has been recently completed by a technique called angle-resolved linear dichroism. By analyzing the fluorescence emission response with respect to the polarization orientation of the exciting light, this technique allows retrieving orientation information of an ensemble of fluorescent molecules, namely the average orientation angle and the amplitude of the angular fluctuations around this average. In this PhD thesis, we implement new methods and instrumentation tools able to improve the robustness and speed of the polarization resolved data analysis, the rate of the data acquisition, and at last to explore the possibility to record molecular 3D orientation information. A scheme able to monitor the real-time orientation properties of fluorescent lipid probes is proposed using a high-speed spinning disk coupled to camera imaging, combined with fast switch- ing of the polarization state by an electro optical modulator. A new data processing method is developed which considerably improves the speed and the precision of the re- trieved information by investigating the sources of bias and uncertainty due to noise and instrumentation factors. The technique has been successfully tested on giant unilamel- lar vesicles and on living cells labeled with different fluorescent lipid probes, DiIC and 18 di-8-ANEPPQ. It was able to acquire precise molecular orientation images at full frame rates in the range of one frame per second. At last in order to probe unambiguously the 3D orientation information of an ensemble of molecules, a new method is proposed and supported by simulations, based on the out-of-plane tuning of the excitation polarization realized in the focusing volume by coherently summing linearly and radially polarized fields. Keywords : fluorescence, polarization, confocal microscopy, spinning disk, orientation distribution, linear dichroism Résumé La microscopie de fluorescence a récemment été complétée par une technique appelée dichroisme linéaire résolu angulairement, basé sur le fait que l’absorption de la lumière est un processus sensible à l’orientation moléculaire. En analysant la réponse d’émission de fluorescence en fonction de l’orientation de la polarisation de la lumière excitatrice, cette technique permet de remonter à l’information d’orientation sur un ensemble de molécules fluorescentes, plus précisément son angle d’orientation moyenne et l’amplitude de ses fluc- tuations angulaires autour de cette moyenne. Dans cette thèse, nous mettons en œuvre de nouvelle méthodes et instrumentations capables d’améliorer la robustesse et la rapidité de l’analyse de données de réponses résolues en polarisation, la vitesse de l’acquisition de données, et d’explorer la possibilité de mesurer l’orientation 3D de molécules. Nous pro- posons une méthode capable de mesurer les propriétés d’orientation de sondes lipidiques fluorescentes par l’utilisation d’un disque de Nipkow couplé à une imagerie par caméra, et combiné avec la modulation rapide de la polarisation par modulateur électro-optique. Une nouvelle méthode de traitement de données est développée pour considérablement améliorer la rapidité et la précision de l’information par une étude des sources de bruit et d’incertitude, dues au bruit et aux facteurs instrumentaux. Cette technique a été testée avec succés sur des vésicules géantes uni-lamellaires et sur cellules vivantes, marquées par les sondes lipidiques DiIC et di-8-ANEPPQ. Cette méthode est capable d’acquérir 18 une information précise sur l’orientation moléculaire à une cadence d’une image par sec- onde. Enfin, afin de sonder de manière non ambiguë l’orientation 3D d’un ensemble de molécules, une nouvelle méthode est proposée, supportée par des simulations numériques, basée sur la variation hors plan de la polarisation d’excitation dans le volume focal par une somme cohérente de champs polarisés linéairement et radialement. Mots clefs : fluorescence, polarisation, microscopie confocale, disque de Nipkow, distri- bution d’orientation, dichroisme linéaire Acknowledgements The work would not have been possible without the help of many people, it is my pleasure to thank them. To the members of the jury: Prof. Markus Sauer, Prof. Colin Sheppard, Dr. Gilles Tessier for their interest in this thesis, the careful reading of the manuscript, having made the journey to Marseille and helpful suggestions. A special acknowledgement to my supervisor Prof. Sophie Brasselet for warm welcome on my study in MOSAIC group and her excellent supervision of this project. Through out this project, she always offered her patience to explain optics and biological problem, encouraged me for our results and inspired me to do open-mind thinking. Moreover, she exhibited her enthusiasm, intuition and efforts to science, which definitely will influence me in my research career. With my special appreciation too, to my co-superviser Dr. Patrick Ferrand for his intel- ligence and patience. He is a genius in setup implementation and programming. During these three years, he gave me a lot of inspiring ideas for setup building and endless helps in detail for Matlab, LabVIEW programming and this project. I am very happy to have been working with him. To Prof. Hervé Rigneault for his great support and helps to my study in the MOSAIC group. To my good partner Alla Kress for her hard works in biology, she made a lot of measure- ments in biological samples, which makes this technique useful. To my good partner Wei He for her implementation test of 3D polarization control setup. To Julien Savatier for his helps in biological problems. To Julien Duboisset for cooperations and discussion on applications of amyloid fibrils. To Cyril Favard for sharing his knowledge of Giant unilamellar vesicles . To Cesar and Fatma-Zohra for their friendly helps to this work and my life, we have spent a lot of nice moments during these three years. To Pascal, Xueqin Chen, Haitham, Sara, Sherazade, Deep, Satish, and all other members of the MOSAIC group for making the nice office atmosphere. At last, I would like to thank my wife for her family supports during these three years. Contents Introduction i 1 Fluorescence is an orientation sensitive process 1 1.1 One-photon fluorescence . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 1.1.1 Absorption and emission spectra properties . . . . . . . . . . . . . . 2 1.1.2 Life time . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4 1.1.3 Absorption and emission transition dipoles . . . . . . . . . . . . . 4 1.2 Fluorescence anisotropy . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7 1.2.1 Fluorescence anisotropy in solution . . . . . . . . . . . . . . . . . . 7 1.2.2 Fluorescence anisotropy in ordered samples . . . . . . . . . . . . . 10 1.3 Linear Dichroism . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13 1.4 Angle resolved linear dichroism . . . . . . . . . . . . . . . . . . . . . . . . 14 1.5 Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16 2 Angle-resolved Linear dichroism (ARLD) 17 2.1 Fluorescence emitted by an ensemble of dipoles . . . . . . . . . . . . . . . 17 2.1.1 Microscopic and macroscopic frame . . . . . . . . . . . . . . . . . . 18 2.1.2 Distribution function . . . . . . . . . . . . . . . . . . . . . . . . . . 20 2.1.3 Emitted fluorescence . . . . . . . . . . . . . . . . . . . . . . . . . . 21 2.2 Setup . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22 2.2.1 Optical setup . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22 2.2.2 Polarization distortions . . . . . . . . . . . . . . . . . . . . . . . . . 24 2.3 Data processing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26 2.3.1 Retrieval of the distribution parameters . . . . . . . . . . . . . . . . 26 2.3.2 Precision analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30 2.3.3 Sources of bias and error criteria . . . . . . . . . . . . . . . . . . . 30 5 CONTENTS 2.4 Example of measurements . . . . . . . . . . . . . . . . . . . . . . . . . . . 33 2.4.1 Giant unilamellar vesicles labeled by DiIC . . . . . . . . . . . . . 33 18 2.4.2 Giant unilamellar vesicles labeled by di-8-ANEPPQ . . . . . . . . . 34 2.4.3 An application in Cos-7 Cells . . . . . . . . . . . . . . . . . . . . . 34 2.5 The limitations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 2.5.1 Out-of-plane orientation η . . . . . . . . . . . . . . . . . . . . . . . 35 2.5.2 Measurement speed . . . . . . . . . . . . . . . . . . . . . . . . . . . 36 2.6 Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 37 3 High frame rate confocal ARLD 43 3.1 Experiment setup . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 44 3.1.1 Parallel confocal imaging . . . . . . . . . . . . . . . . . . . . . . . . 44 3.1.2 Optical setup . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 47 3.1.3 Polarization control . . . . . . . . . . . . . . . . . . . . . . . . . . . 48 3.1.4 Hardware . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 50 3.2 Calibrations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 51 3.2.1 Polarization Calibration . . . . . . . . . . . . . . . . . . . . . . . . 51 3.2.2 Camera calibration . . . . . . . . . . . . . . . . . . . . . . . . . . . 54 3.2.3 Data processing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 56 3.2.4 Precision analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . 58 3.3 Examples of measurements . . . . . . . . . . . . . . . . . . . . . . . . . . . 59 3.3.1 Giant unilamellar vesicles labeled with DiIC . . . . . . . . . . . . 59 18 3.3.2 Cos-7 cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 63 3.4 Acquisition rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 65 3.5 Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 65 4 Determining 3D orientation distribution 67 4.1 Measuring of 3D orientation: the case of a single dipole . . . . . . . . . . . 67 4.2 3D excitation and detection for a distribution of dipoles . . . . . . . . . . . 72 4.2.1 About the need of a full calculation . . . . . . . . . . . . . . . . . 72 4.2.2 The electric field in the focus volume of a high NA objective . . . . 74 4.2.3 Emission field by an excited dipole . . . . . . . . . . . . . . . . . . 79 4.2.4 Image of a dipole and detection efficiency . . . . . . . . . . . . . . . 82 4.3 Proposed method to probe the order in 3D . . . . . . . . . . . . . . . . . . 87 4.3.1 Scanning scheme of the polarization of the exciting field . . . . . . . 87 6 CONTENTS 4.3.2 Unambiguous retrieval of (η,ψ) . . . . . . . . . . . . . . . . . . . . 90 4.3.3 The membrane geometry . . . . . . . . . . . . . . . . . . . . . . . . 93 4.3.4 Practical setup . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 96 4.4 Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 97 Conclusion and outlooks 98 A The effect of Homo-FRET on polarization resolved measurements 101 A.1 Modeling the fluorescence response of molecule in a cone with Homo-FRET 102 A.1.1 Definition of the coupled dipoles . . . . . . . . . . . . . . . . . . . . 102 A.1.2 Absorption probability of the donor . . . . . . . . . . . . . . . . . . 104 A.1.3 Detection probability of the acceptor . . . . . . . . . . . . . . . . . 104 A.1.4 Coupling factor . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 105 A.1.5 The emitted fluorescence . . . . . . . . . . . . . . . . . . . . . . . . 105 A.2 Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 106 A.2.1 Cones distributed in the whole focal volume . . . . . . . . . . . . . 106 A.2.2 Analysis for cones models distributed in membranes . . . . . . . . . 108 A.3 Brief analysis for FRET . . . . . . . . . . . . . . . . . . . . . . . . . . . . 110 B Measurement of a polarization state and analysis of polarization distor- tions 113 B.1 Measurement of a polarization state . . . . . . . . . . . . . . . . . . . . . . 113 B.1.1 Characterization of a polarization state . . . . . . . . . . . . . . . . 113 B.1.2 The quarter waveplate method . . . . . . . . . . . . . . . . . . . . . 113 B.1.3 Parameters retrieval . . . . . . . . . . . . . . . . . . . . . . . . . . 116 B.2 Analysis of polarization distortions induced by an optical path . . . . . . 117 C Preparation of samples 119 C.1 Preparation of Giant Unilamellar Vesicles (GUVs) . . . . . . . . . . . . . . 119 C.2 Preparation of cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 119 Bibliography 121 7 CONTENTS 8 Introduction Molecular structural organization is a crucial factor in biological processes where func- tions can be closely related to the orientation distribution of molecules. For instance, in cell biology the structural organization of proteins and lipids are the direct signature of molecular interaction mechanisms which play a determining role in biological functions such as cell shape maintenance and motility [1], vesicular trafficking [2], and signaling [3]. Polarization as one of the property of light has been exploited for studying the ori- entation related information of molecules. In 1920, F. Weigert firstly discovered that the fluorescencefromsolutionsofdyesispolarized[4]. Basedonthisproperty,amethodcalled fluorescenceanisotropywasproposedbycomparingthefluorescencesignalsrecordedalong analysis directions parallel and perpendicular to the incident polarization. Because of the sensitivity to depolarization process, it has been primarily used for decades in studies of solution in order to address issues such as rotational diffusion and energy transfer be- tween neighbor molecules. Later, this technique has been introduced to investigate the organization of biomolecular assemblies in ordered media such as lipid membranes [5, 6]. Another technique, called linear dichroism (LD) was also proposed based on the fact that the absorption probability is determined by the relative orientation between the absorbing dipole and exciting electric field. This method is now widely used to retrieve molecules orientation information, by comparing the fluorescence response recorded for two orthog- onal directions of polarization of excitation. Compared to fluorescence anisotropy, the LD is not affected by depolarization processes such as fast rotational diffusion or energy transfer [7, 8]. During the past ten years, an increasing number of research groups have dedicated their studies to LD techniques to explore biological samples such as amyloids [8, 9], septins [6], membranes [7], actin [10]. In ordered assemblies however, which can be characterized by a more complex set of parameters, these two methods that utilize two orthogonal directions of measurements, suffer from some limitations. Indeed the ratio between two measurements supplies only i

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Keywords : fluorescence, polarization, confocal microscopy, spinning disk, orientation distribution, linear .. the absorption gets maximum. On the
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