TY - CHAP
T1 - Correlative light-electron microscopy as a tool to study in vivo dynamics and ultrastructure of intracellular structures
AU - Polishchuk, Elena V.
AU - Polishchuk, Roman S.
AU - Luini, Alberto
PY - 2012
Y1 - 2012
N2 - Correlative light-electron microscopy (CLEM) is a very effective technique that combines live-cell imaging and immuno-electron microscopy for ultrastructural morphological characterization of dynamic intracellular organelles. The use of green fluorescent protein (GFP)-tagged chimeras allows the user to follow the movements and/or behavior of intracellular structures in a live cell and to fix it at the moment of interest. The subsequent immuno-electron microscopy processing can then reveal the three-dimensional architecture of the same structure, together with precise recognition of the GFP-labeled protein. The process resembles the taking of a high-resolution snapshot of an interesting live scene. Considering that CLEM is a very useful but technically demanding and time-consuming technique, accurate protocols will be helpful to simplify the work of scientists who are willing to apply this method for their own purposes. Here, we present a detailed protocol that describes all of the "tricks" and know-hows involved in carrying out the crucial steps of a CLEM experiment
AB - Correlative light-electron microscopy (CLEM) is a very effective technique that combines live-cell imaging and immuno-electron microscopy for ultrastructural morphological characterization of dynamic intracellular organelles. The use of green fluorescent protein (GFP)-tagged chimeras allows the user to follow the movements and/or behavior of intracellular structures in a live cell and to fix it at the moment of interest. The subsequent immuno-electron microscopy processing can then reveal the three-dimensional architecture of the same structure, together with precise recognition of the GFP-labeled protein. The process resembles the taking of a high-resolution snapshot of an interesting live scene. Considering that CLEM is a very useful but technically demanding and time-consuming technique, accurate protocols will be helpful to simplify the work of scientists who are willing to apply this method for their own purposes. Here, we present a detailed protocol that describes all of the "tricks" and know-hows involved in carrying out the crucial steps of a CLEM experiment
KW - Correlative light-electron microscopy
KW - Gold enhancement
KW - Green fluorescent protein
KW - Gridded coverslips
KW - Immuno-electron microscopy
KW - Live-cell imaging
KW - Paraformaldehyde-glutaraldehyde fixation
KW - Resin embedding
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U2 - 10.1007/978-1-62703-56-4_20
DO - 10.1007/978-1-62703-56-4_20
M3 - Chapter
AN - SCOPUS:84934436107
SN - 9781627030557
VL - 931
T3 - Methods in Molecular Biology
SP - 413
EP - 422
BT - Methods in Molecular Biology
PB - Humana Press Inc.
ER -