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3D reconstruction of neural circuits from serial EM images [Elektronische Ressource] / vorgelegt von Nina Maack

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3D Reconstruction of Neural Circuitsfrom Serial EM ImagesNina MaackMunchen 20083D Reconstruction of Neural Circuitsfrom Serial EM ImagesNina MaackDissertationzur Erlangung des Doktorgradesder Naturwissenschaften (Dr. rer. nat.)der Fakult at fur Biologieder Ludwig{Maximilians{Universit at Munc henAngefertigt am Max-Planck-Institut fur NeurobiologieAbteilung "Neuronale Informationsverarbeitung"vorgelegt vonNina Maackaus Landsberg/LechMunc hen, den 29.04.2008Erstgutachter: Professor Dr. Alexander BorstZweitgutachter: Professor Dr. Benedikt GrotheTag der mundlic hen Prufung: 20.06.2008Table of ContentsTable of Contents iList of gures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . iiiAbbreviations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ivAbstract 11 Introduction 31.1 Neural Circuit Reconstruction . . . . . . . . . . . . . . . . . . . . . . . . . 31.2 Microscopy Techniques . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 51.2.1 Serial Section Transmission Electron Microscopy . . . . . . . . . . . 51.2.2 Serial Block-Face Scanning Electrony . . . . . . . . . . 71.3 Goals . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 122 Materials and Methods 132.1 Specimen Preparation for SBFSEM . . . . . . . . . . . . . . . . . . . . . . 132.1.1 Fly Brain Preparation and Fixation . . . . . . . . . . . . . . . . . . 132.1.2 Staining . . . . . . . . . .

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3D Reconstruction of Neural Circuits
from Serial EM Images
Nina Maack
Munchen 20083D Reconstruction of Neural Circuits
from Serial EM Images
Nina Maack
Dissertation
zur Erlangung des Doktorgrades
der Naturwissenschaften (Dr. rer. nat.)
der Fakult at fur Biologie
der Ludwig{Maximilians{Universit at Munc hen
Angefertigt am Max-Planck-Institut fur Neurobiologie
Abteilung "Neuronale Informationsverarbeitung"
vorgelegt von
Nina Maack
aus Landsberg/Lech
Munc hen, den 29.04.2008Erstgutachter: Professor Dr. Alexander Borst
Zweitgutachter: Professor Dr. Benedikt Grothe
Tag der mundlic hen Prufung: 20.06.2008Table of Contents
Table of Contents i
List of gures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . iii
Abbreviations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . iv
Abstract 1
1 Introduction 3
1.1 Neural Circuit Reconstruction . . . . . . . . . . . . . . . . . . . . . . . . . 3
1.2 Microscopy Techniques . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
1.2.1 Serial Section Transmission Electron Microscopy . . . . . . . . . . . 5
1.2.2 Serial Block-Face Scanning Electrony . . . . . . . . . . 7
1.3 Goals . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12
2 Materials and Methods 13
2.1 Specimen Preparation for SBFSEM . . . . . . . . . . . . . . . . . . . . . . 13
2.1.1 Fly Brain Preparation and Fixation . . . . . . . . . . . . . . . . . . 13
2.1.2 Staining . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13
2.1.3 Final Preparation of SBFSEM Sample . . . . . . . . . . . . . . . . 14
2.1.4 Microscopy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14
2.2 Software Development Tools . . . . . . . . . . . . . . . . . . . . . . . . . . 15
2.2.1 Hardware . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15
2.2.2 Programming Languages . . . . . . . . . . . . . . . . . . . . . . . . 16
3 Results 17
3.1 SBFSEM Data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17
3.2 Software - Neuron2D . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18
3.2.1 Image Registration . . . . . . . . . . . . . . . . . . . . . . . . . . . 19
3.2.1.1 Tiling Technique of SBFSEM . . . . . . . . . . . . . . . . 19ii Table of Contents
3.2.1.2 Overview of Image Registration Methods . . . . . . . . . . 20
3.2.1.3 Implementation - Image Registration . . . . . . . . . . . . 22
3.2.1.4 Results - Image Registration . . . . . . . . . . . . . . . . . 22
3.2.2 Image Preprocessing . . . . . . . . . . . . . . . . . . . . . . . . . . 24
3.2.2.1 Overview of Image Filtering Algorithms . . . . . . . . . . 24
3.2.2.2 Implementation - Image Preprocessing . . . . . . . . . . . 27
3.2.2.3 Results - Image Preprocessing . . . . . . . . . . . . . . . . 28
3.2.3 Image Segmentation . . . . . . . . . . . . . . . . . . . . . . . . . . 29
3.2.3.1 Contour-Propagation Algorithms . . . . . . . . . . . . . . 30
3.2.3.2 Canny Segmentation Algorithm . . . . . . . . . . . . . . . 38
3.2.3.3 Manual Interaction . . . . . . . . . . . . . . . . . . . . . 41
3.2.3.4 Quanti cation of Segmentation Algorithms . . . . . . . . . 43
3.2.3.5 Data Storage . . . . . . . . . . . . . . . . . . . . . . . . . 47
3.3 Software - Neuron3D . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 49
3.3.1 Surface Reconstruction . . . . . . . . . . . . . . . . . . . . . . . . . 49
3.3.1.1 Overview of Surface Reconstruction Algorithms . . . . . . 49
3.3.1.2 Implementation - Surface Reconstruction . . . . . . . . . 51
3.3.1.3 Integration of Branches . . . . . . . . . . . . . . . . . . . 53
3.3.2 3D Image Editing . . . . . . . . . . . . . . . . . . . . . . . . . . . . 56
3.3.2.1 Surface Representation . . . . . . . . . . . . . . . . . . . . 56
3.3.2.2 Lighting Implementation . . . . . . . . . . . . . . . . . . 57
3.3.2.3 Integration of SBFSEM Data . . . . . . . . . . . . . . . . 61
3.3.3 Results and Data Storage . . . . . . . . . . . . . . . . . . . . . . . 62
3.4 Software Usage and Evaluation . . . . . . . . . . . . . . . . . . . . . . . . 63
4 Discussion 69
4.1 Software Neuron2D and Neuron3D . . . . . . . . . . . . . . . . . . . . . . 69
4.2 Biological Application of the Software . . . . . . . . . . . . . . . . . . . . . 71
4.3 Outlook . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 74
5 References 75
Acknowledgements 83
Curriculum Vitae 85iii
List of Figures
1.1 Visual system of the y . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4
1.2 Technique of SSTEM and TEM . . . . . . . . . . . . . . . . . . . . . . . . 6
1.3 Schematic of SEM and BSE coe cient . . . . . . . . . . . . . . . . . . . . 8
1.4 Schematic of ESEM . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9
1.5 Schematic of SBFSEM and working principle . . . . . . . . . . . . . . . . . 10
3.1 Example SBFSEM image stack . . . . . . . . . . . . . . . . . . . . . . . . 18
3.2 tiling SBFSEM . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19
3.3 Results image registration . . . . . . . . . . . . . . . . . . . . . . . . . . . 24
3.4 Example image preprocessing . . . . . . . . . . . . . . . . . . . . . . . . . 28
3.5 Di erent gray value distributions of SBFSEM image stacks . . . . . . . . . 30
3.6 Splitting of objects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31
3.7 Schematic illustration of the algorithm . . . . . . . . . . . . . . . . . . . . 34
3.8 Segmentation of the rst image (screen shots) . . . . . . . . . . . . . . . . 38
3.9 Example Canny segmentation algorithm . . . . . . . . . . . . . . . . . . . 41
3.10 Quanti cation of performance . . . . . . . . . . . . . . . . . . . . . . . . . 44
3.11 Typical errors made by the algorithm . . . . . . . . . . . . . . . . . . . . . 45
3.12 Test Canny segmentation . . . . . . . . . . . . . . . . . . . . . . . . . . . . 46
3.13 Typical errors made by the Canny segmentation algorithm . . . . . . . . . 46
3.14 Example XML output le . . . . . . . . . . . . . . . . . . . . . . . . . . . 48
3.15 Surface reconstruction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 53
3.16 Branched contours . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 55
3.17 Surface simpli cation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 58
3.18 Shadow implementation . . . . . . . . . . . . . . . . . . . . . . . . . . . . 61
3.19 Reconstruction of axonal structures . . . . . . . . . . . . . . . . . . . . . . 63
3.20 Selected test regions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 65
3.21 Software evaluation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 67
4.1 Visualization of synapses with SBFSEM . . . . . . . . . . . . . . . . . . . 71
4.2 Fluorescein and neurobiotin staining of VS-cells . . . . . . . . . . . . . . . 72
4.3 EM images of gap junctions . . . . . . . . . . . . . . . . . . . . . . . . . . 73iv
Abbreviations
2D Two-Dimensional
3D Three-Dimensional
API Application Programming Interface
BSE Backscattered Electrons
DTW Dynamic Time Warping
EM Electron Microscope
EMD Elementary Motion Detector
ESEM Environmental Scanning Electron Microscope
GUI Graphical User Interface
LMCs Large Monopolar Cells
LPTCs Lobula Plate Tangential Cells
NLDF Nonlinear Di usion Filter
NP Non-deterministic Polynomial time
OpenGL Open Graphics Library
PB Phosphate Bu er
RAM Random Access Memory
SBFSEM Serial Block-Face Scanning Electron Microscopy
SDF Signed Distance Function
SE Secondary Electrons
SEM Scanning Electron Microscopy
TEM Transmission Electron Microscopy
XML Extensible Markup Language