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A Fluorescent Speckle Microscopy Study

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A Fluorescent Speckle Microscopy Study Research Collection Doctoral Thesis High-resolution analysis of F-actin meshwork kinetics and kinematics using computational Fluorescent Speckle Microscopy Author(s): Ponti, Aaron Christian Publication Date: 2003 Permanent Link: https://doi.org/10.3929/ethz-a-004650398 Rights / License: In Copyright - Non-Commercial Use Permitted This page was generated automatically upon download from the ETH Zurich Research Collection. For more information please consult the Terms of use. ETH Library DISS. ETH NO. 15286 High-Resolution Analysis of F-Actin Meshwork Kinetics and Kinematics using Computational Fluorescent Speckle Microscopy A dissertation submitted to the SWISS FEDERAL INSTITUTE OF TECHNOLOGY ZURICH For the degree of Doctor of Sciences Presented by Aaron Christian Ponti Dipl. Natw. ETHZ Born 30.10.1975 Citizen of Breganzona (TI) Accepted on the recommendation of Prof. Dr. Gaudenz Danuser, examiner Prof. Dr. Jonathon Howard, co-examiner Prof. Dr. Yves Barral, co-examiner 2003 2 Table of contents Table of contents TABLE OF CONTENTS...................................................................................................................................... 3 SUMMARY ........................................................................................................................................................... 7 SOMMARIO ......................................................................................................................................................... 9 1 INTRODUCTION .......................................................................................................................................... 13 1.1 FUNDAMENTS OF CELL MOTILITY ............................................................................................................... 14 1.1.1 Cell locomotion ..................................................................................................................................... 14 1.1.2 Actin ...................................................................................................................................................... 15 1.1.3 Protrusion is driven by actin-polymerization........................................................................................ 16 1.1.4 The role of signaling on the spatial control of actin assembly.............................................................. 18 1.1.5 Stabilization of the actin meshwork....................................................................................................... 20 1.1.6 Cell body translocation ......................................................................................................................... 20 1.2 FLUORESCENT SPECKLE MICROSCOPY ........................................................................................................ 22 1.2.1 FSM origins and fields of application................................................................................................... 22 1.2.2 Quantitative FSM.................................................................................................................................. 25 1.3 OUTLINE OF THE THESIS ............................................................................................................................. 25 2 COMPUTATIONAL ANALYSIS OF F-ACTIN TURNOVER IN CORTICAL ACIN MESHWORKS USING FLUORESCENT SPECKLE MICROSCOPY .............................................................................. 27 2.1 INTRODUCTION........................................................................................................................................... 28 2.2 MATERIALS AND METHODS ........................................................................................................................ 29 2.2.1 Software development ........................................................................................................................... 29 2.2.2 Synthesis of FSM movies using Monte Carlo simulations of dynamic F-actin meshworks................... 29 2.2.3 Cell Manipulations................................................................................................................................ 31 2.2.4 Time-lapse Spinning Disk Confocal FSM ............................................................................................. 32 2.2.5 Characterization of image noise ........................................................................................................... 32 2.2.5.1 Titration experiments........................................................................................................................................ 32 2.2.5.2 Calibration of a noise model............................................................................................................................. 33 2.3 ALGORITHM ............................................................................................................................................... 33 2.3.1 Noise reduction and local maximum selection...................................................................................... 33 2.3.2 Statistical selection of speckles ............................................................................................................. 36 2.3.3 Micro-movement tracking ..................................................................................................................... 40 2.3.4 Removal of time gaps in speckle trajectories ........................................................................................ 41 2.3.5 Classification of birth and death events................................................................................................ 42 2.3.6 Kinetic analysis of birth and death events and accumulation of actin turnover maps .......................... 45 2.4 RESULTS..................................................................................................................................................... 48 2.4.1 Verification of the algorithm on synthetic data..................................................................................... 48 2.4.2 FSM analysis of cortical actin meshworks reveals cyclic polymer turnover ........................................ 49 3 Table of contents 2.4.3 Latrunculin A perfusion induces a spreading of the cycle frequencies ................................................. 53 2.5 DISCUSSION................................................................................................................................................ 54 2.5.1 FSM analysis relies on a statistical separation of significant events.................................................... 54 2.5.2 The effect of bleaching on FSM analysis is minimal and can be eliminated completely by an appropriate correction scheme........................................................................................................................ 56 2.5.3 The effect of focus changes.................................................................................................................... 59 2.5.4 Local meshwork contractions are most likely to result in ghost speckles which are excluded from the analysis............................................................................................................................................................ 61 2.6 ACKNOWLEDGEMENTS ............................................................................................................................... 63 3 RECOVERY, VISUALIZATION, AND ANALYSIS OF ACTIN AND TUBULIN POLYMER FLOW IN LIVE CELLS: A FLUORESCENT SPECKLE MICROSCOPY STUDY ......................................... 65 3.1 INTRODUCTION........................................................................................................................................... 66 3.1.1 FSM of filamentous actin flow at the leading edge of migrating cells .................................................. 67 3.1.2 FSM of poleward microtubule flux in mitotic spindles.......................................................................... 68 3.1.3 Scope of the paper................................................................................................................................. 69 3.2 MATERIALS AND METHODS ........................................................................................................................ 70 3.2.1 FSM of actin in migrating newt lung epithelial cells ............................................................................70 3.2.2 FSM of mitotic spindle in Xenopus eggs extracts.................................................................................. 70 3.3 ALGORITHM ............................................................................................................................................... 71 3.3.1 Signal preconditioning.......................................................................................................................... 71 3.3.2 Particle generation................................................................................................................................ 71 3.3.3 Particle selection................................................................................................................................... 71 3.3.4 Generation of candidate matches.......................................................................................................... 72 3.3.5 Selection of matches.............................................................................................................................
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