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Streamlined Embedding of Cell Monolayers on Gridded Glass-Bottom Imaging Dishes for Correlative Light and Electron Microscopy

Published online by Cambridge University Press:  20 October 2010

Hugo H. Hanson ,
James E. Reilly ,
Rebecca Lee ,
William G. Janssen  and
Greg R. Phillips
Hugo H. Hanson
Affiliation:
Department of Neuroscience, Mount Sinai School of Medicine, One Gustave L. Levy Place, New York, NY 10029, USA
James E. Reilly
Affiliation:
Department of Neuroscience, Mount Sinai School of Medicine, One Gustave L. Levy Place, New York, NY 10029, USA
Rebecca Lee
Affiliation:
Department of Neuroscience, Mount Sinai School of Medicine, One Gustave L. Levy Place, New York, NY 10029, USA
William G. Janssen
Affiliation:
Department of Neuroscience, Mount Sinai School of Medicine, One Gustave L. Levy Place, New York, NY 10029, USA
Greg R. Phillips*
Affiliation:
Department of Neuroscience, Mount Sinai School of Medicine, One Gustave L. Levy Place, New York, NY 10029, USA
*
Corresponding author. E-mail: [email protected]
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Abstract

Correlative light and electron microscopy (CLEM) has facilitated study of intracellular trafficking. Routine application of CLEM would be advantageous for many laboratories but previously described techniques are particularly demanding, even for those with access to laser scanning confocal microscopy (LSCM) and transmission electron microscopy (TEM). We describe streamlined methods for TEM of green fluorescent protein (GFP)-labeled organelles after imaging by LSCM using gridded glass bottom imaging dishes. GFP-MAP 1A/1B LC3 (GFP-LC3) transfected cells were treated with rapamycin, fixed and imaged by LSCM. Confocal image stacks were acquired enabling full visualization of each GFP-LC3 labeled organelle. After LSCM, cells were embedded for TEM using a simplified two step method that stabilizes the glass bottom such that the block can be separated from the glass by mild heating. All imaging and TEM processing are performed in the same dish. The LSCM imaged cells were relocated on the block and serial sectioned. Correlation of LSCM, DIC, and TEM images was facilitated by cellular landmarks. All GFP labeled structures were successfully reidentified and imaged by serial section TEM. This method could make CLEM more accessible to nonspecialized laboratories with basic electron microscopy expertise and could be used routinely to confirm organelle localization of fluorescent puncta.

Keywords

organellegreen fluorescent proteinlive imagingautophagosomevesicletrafficking
Type
Fluorescence and Confocal Microscopies
Information
Microscopy and Microanalysis , Volume 16 , Issue 6 , December 2010 , pp. 747 - 754
Copyright
Copyright © Microscopy Society of America 2010

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