From live-cell imaging to scanning electron microscopy (SEM): the use of green fluorescent protein (GFP) as a common label.

2.50
Hdl Handle:
http://hdl.handle.net/10541/55827
Title:
From live-cell imaging to scanning electron microscopy (SEM): the use of green fluorescent protein (GFP) as a common label.
Authors:
Drummond, Sheona P; Allen, Terence D
Abstract:
The identification and characterization of many biological substructures at high resolution requires the use of electron microscopy (EM) technologies. Scanning electron microscopy (SEM) allows the resolution of cellular structures to approximately 3 nm and has facilitated the direct visualization of macromolecular structures, such as nuclear pore complexes (NPCs), which are essential for nucleo-cytoplasmic molecular trafficking. However, SEM generates only static images of fixed samples and therefore cannot give unambiguous information about protein dynamics. The investigation of active processes and analysis of protein dynamics has greatly benefited from the development of molecular biology techniques whereby vectors can be generated and transfected into tissue culture cells for the expression of specific proteins tagged with a fluorescent moiety for real-time light microscopy visualization. As light microscopy is limited in its powers of resolution relative to electron microscopy, it has been important to adapt a protocol for the processing of samples for real-time imaging by conventional light microscopy with protein labels that can also be identified by SEM. This allows correlation of dynamic events with high resolution molecular and structural identification. This method describes the use of GFP for tracking the dynamic distribution of NPC components in real-time throughout the cell cycle and for high resolution immuno-SEM labeling to determine localization at the nanometer level.
Affiliation:
Department of Structural Cell Biology, Paterson Institute for Cancer Research, University of Manchester, Manchester M20 4BX, United Kingdom.
Citation:
From live-cell imaging to scanning electron microscopy (SEM): the use of green fluorescent protein (GFP) as a common label. 2008, 88:97-108 Methods Cell Biol.
Journal:
Methods in Cell Biology
Issue Date:
2008
URI:
http://hdl.handle.net/10541/55827
DOI:
10.1016/S0091-679X(08)00406-8
PubMed ID:
18617030
Type:
Book chapter
Language:
en
ISSN:
0091-679X
Appears in Collections:
All Paterson Institute for Cancer Research

Full metadata record

DC FieldValue Language
dc.contributor.authorDrummond, Sheona P-
dc.contributor.authorAllen, Terence D-
dc.date.accessioned2009-03-16T17:58:50Z-
dc.date.available2009-03-16T17:58:50Z-
dc.date.issued2008-
dc.identifier.citationFrom live-cell imaging to scanning electron microscopy (SEM): the use of green fluorescent protein (GFP) as a common label. 2008, 88:97-108 Methods Cell Biol.en
dc.identifier.issn0091-679X-
dc.identifier.pmid18617030-
dc.identifier.doi10.1016/S0091-679X(08)00406-8-
dc.identifier.urihttp://hdl.handle.net/10541/55827-
dc.description.abstractThe identification and characterization of many biological substructures at high resolution requires the use of electron microscopy (EM) technologies. Scanning electron microscopy (SEM) allows the resolution of cellular structures to approximately 3 nm and has facilitated the direct visualization of macromolecular structures, such as nuclear pore complexes (NPCs), which are essential for nucleo-cytoplasmic molecular trafficking. However, SEM generates only static images of fixed samples and therefore cannot give unambiguous information about protein dynamics. The investigation of active processes and analysis of protein dynamics has greatly benefited from the development of molecular biology techniques whereby vectors can be generated and transfected into tissue culture cells for the expression of specific proteins tagged with a fluorescent moiety for real-time light microscopy visualization. As light microscopy is limited in its powers of resolution relative to electron microscopy, it has been important to adapt a protocol for the processing of samples for real-time imaging by conventional light microscopy with protein labels that can also be identified by SEM. This allows correlation of dynamic events with high resolution molecular and structural identification. This method describes the use of GFP for tracking the dynamic distribution of NPC components in real-time throughout the cell cycle and for high resolution immuno-SEM labeling to determine localization at the nanometer level.en
dc.language.isoenen
dc.subjectLive-Cell Imagingen
dc.subjectElectron Microscopyen
dc.subjectSEMen
dc.subjectGFPen
dc.subject.meshAnimals-
dc.subject.meshCell Culture Techniques-
dc.subject.meshCell Physiological Phenomena-
dc.subject.meshGreen Fluorescent Proteins-
dc.subject.meshHumans-
dc.subject.meshImmunohistochemistry-
dc.subject.meshMicroscopy, Electron, Scanning-
dc.subject.meshModels, Biological-
dc.subject.meshStaining and Labeling-
dc.subject.meshTransfection-
dc.titleFrom live-cell imaging to scanning electron microscopy (SEM): the use of green fluorescent protein (GFP) as a common label.en
dc.typeBook chapteren
dc.contributor.departmentDepartment of Structural Cell Biology, Paterson Institute for Cancer Research, University of Manchester, Manchester M20 4BX, United Kingdom.en
dc.identifier.journalMethods in Cell Biologyen

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