UID:
edoccha_9958132053302883
Umfang:
1 online resource (493 p.)
ISBN:
0-12-801321-4
Serie:
Methods in Cell Biology, Volume 124
Inhalt:
This new volume of Methods in Cell Biology looks at methods for analyzing correlative light and electron microscopy (CLEM). With CLEM, people try to combine the advantages of both worlds, i.e. the dynamics information obtained by light microscopy and the ultrastructure as provided by electron microscopy. This volume contains the latest techniques on correlative microscopy showing that combining two imaging modalities provides more than each technique alone. Most importantly it includes the essential protocols, including tips, tricks and images for you to repeat these exciting techniques in you
Anmerkung:
Description based upon print version of record.
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Front Cover; Contents; Contributors; Preface; Acknowledgments; Chapter 1: Retracing in Correlative Light Electron Microscopy: Where is My Object of Interest?; Abstract; Introduction; 1.1. Steps in the CLEM Workflow; 1.1.1. Probes; 1.1.2. Processing; 1.1.3. Analysis; 1.2. Methods; 1.2.1. Cell culture and immunostaining; 1.2.2. Light microscopy imaging; 1.2.3. Sample processing for EM-Coarse alignment; 1.2.4. Automated alignment; 1.3. Materials; Conclusions; References; Chapter 2: Fluorescing the Electron: Strategies in Correlative Experimental Design; Abstract; Introduction
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2.1. Strategies for CLEM2.1.1. Live-cell imaging and time-resolved correlation between light and electron microscopy; 2.1.2. The needle in a haystack-Identification of transfected cells; 2.1.3. High-resolution CLEM for precise alignment of fluorescence and EM Data; 2.1.4. Staining for TEM; 2.2. Methods; 2.2.1. Cryo-immobilization and segmentation of microtubules in mitotic spindles in C. elegans embryos by electron tomography; 2.2.2. Localization of EGFP-Sec22-transfected neuron cells; 2.2.3. High-resolution CLEM: Sample preparation, sectioning, image acquisition, and alignment
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2.2.3.1. Sectioning and fiducial application2.2.3.2. Fluorescence microscopy; 2.2.3.3. TEM imaging; 2.2.3.4. High-resolution CLEM image alignment; 2.3. Materials; 2.3.1. Reagents; 2.3.2. Cell cultures; 2.3.3. Light microscopy; 2.3.4. Electron microscopy and image processing; 2.4. Discussion; 2.4.1. Toward super-resolution CLEM; Acknowledgments; References; Chapter 3: Metallothioneins for Correlative Light and Electron Microscopy; Abstract; Introduction; 3.2. Methods; 3.2.1. General technical considerations; 3.2.2. MT tagging for bacteria; 3.2.3. Mammalian cell lines; 3.2.4. Yeast
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3.2.5. A general protocol: How to get started3.3. Discussion; 3.3.1. Resolution and sensitivity; 3.3.2. Multiple labeling; 3.3.3. New protocols for CLEM; 3.3.4. MT for three-dimensional electron microscopy; Acknowledgments; References; Chapter 4: Correlative Light Microscopy and Electron Tomography to Study Von Willebrand Factor Exocytosis from Vascular Endot; Abstract; Introduction; 4.1. Rationale; 4.2. Methods; 4.2.1. Cell culture; 4.2.2. Regulated WPB exocytosis; 4.2.3. Fluorescent labeling; 4.2.4. Confocal microscopy; 4.2.5. Sample preparation for EM; 4.2.6. Correlating LM to TEM sections
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4.2.7. Serial tomography of correlating structures4.2.8. Reconstruction and joining of serial tomograms; 4.2.9. Modeling and visualization; 4.3. Materials and Instrumentation; 4.3.1. Cell culture; 4.3.2. WPB exocytosis and fixation; 4.3.3. Fluorescence labeling; 4.3.4. Light microscopy; 4.3.5. Electron microscopy; 4.3.6. Data processing; 4.4. Discussion; 4.5. Outlook; Acknowledgments; References; Chapter 5: A Simple Procedure to Analyze Positions of Interest in Infectious Cell Cultures by Correlative Light and Electron ; Abstract; Introduction and Rationale; 5.1. Methods
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5.1.1. Cell culture, infection and transfection
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English
Weitere Ausg.:
ISBN 0-12-801075-4
Sprache:
Englisch
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