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Confocal/TEM Overlay Microscopy: A Simple Method for Correlating Confocal and Electron Microscopy of Cells Expressing GFP/YFP Fusion Proteins

Published online by Cambridge University Press:  04 July 2008

Douglas R. Keene ,
Sara F. Tufa ,
Gregory P. Lunstrum ,
Paul Holden  and
William A. Horton
Douglas R. Keene*
Affiliation:
Research Department, Shriners Hospital for Children, Portland, OR 97239, USA Oregon Health Sciences University, Portland, OR 97239, USA
Sara F. Tufa
Affiliation:
Research Department, Shriners Hospital for Children, Portland, OR 97239, USA
Gregory P. Lunstrum
Affiliation:
Research Department, Shriners Hospital for Children, Portland, OR 97239, USA
Paul Holden
Affiliation:
Research Department, Shriners Hospital for Children, Portland, OR 97239, USA
William A. Horton
Affiliation:
Research Department, Shriners Hospital for Children, Portland, OR 97239, USA Oregon Health Sciences University, Portland, OR 97239, USA
*
Corresponding author. E-mail: [email protected]
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Abstract

Genetic manipulation allows simultaneous expression of green fluorescent protein (GFP) and its derivatives with a wide variety of cellular proteins in a variety of living systems. Epifluorescent and confocal laser scanning microscopy (confocal) localization of GFP constructs within living tissue and cell cultures has become routine, but correlation of light microscopy and high resolution transmission electron microscopy (TEM) on components within identical cells has been problematic. In this study, we describe an approach that specifically localizes the position of GFP/yellow fluorescent protein (YFP) constructs within the same cultured cell imaged in the confocal and transmission electron microscopes. We present a simplified method for delivering cell cultures expressing fluorescent fusion proteins into LR White embedding media, which allows excellent GFP/YFP detection and also high-resolution imaging in the TEM. Confocal images from 0.5-μm-thick sections are overlaid atop TEM images of the same cells collected from the next serial ultrathin section. The overlay is achieved in Adobe Photoshop by making the confocal image somewhat transparent, then carefully aligning features within the confocal image over the same features visible in the TEM image. The method requires no specialized specimen preparation equipment; specimens are taken from live cultures to embedding within 8 h, and confocal transmission overlay microscopy can be completed within a few hours.

Keywords

confocal transmission overlay (CTO) microscopycorrelative microscopytransmission electron microscopyAdobe Photoshopcell culturetechniquemethod
Type
Biological Applications
Information
Microscopy and Microanalysis , Volume 14 , Issue 4 , August 2008 , pp. 342 - 348
Copyright
Copyright © Microscopy Society of America 2008

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