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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: drk@shcc.org
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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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References

REFERENCES

Biel, S.S., Kawaschinski, K., Wittern, K.-P., Hintze, U. & Wepf, R. (2003). From tissue to cellular ultrastructure: Closing the gap between micro- and nanostructural imaging. J Microsc 212, 91–99.CrossRefGoogle Scholar
Brorson, S.-H. (1998). Antigen detection on resin sections and methods for improving the immunogold labeling by manipulating the resin. Histol Histopathol 13, 275–281.Google ScholarPubMed
Cho, J.Y., Guo, C., Torello, M., Lunstrum, G.P., Iwata, T., Deng, C. & Horton, W.A. (2004). Defective lysosomal targeting of activated fibroblast growth factor receptor 3 in achondroplasia. Proc Natl Acad Sci 101, 609–614.CrossRefGoogle ScholarPubMed
Grabenbauer, M., Geerts, W.J., Fernandez-Rodriguez, J., Hoenger, A., Koster, A.J. & Nilsson, T. (2005). Correlative microscopy and electron tomography of GFP through photooxidation. Nat Methods 2, 857–862.CrossRefGoogle ScholarPubMed
Groos, S., Reale, E. & Luciano, L. (2001). Re-evaluation of epoxy resin sections for light and electron microscopic immunostaining. J Histochem Cytochem 49, 397–406.CrossRefGoogle ScholarPubMed
Holden, P., Keene, D.R., Lunstrum, G.P., Bächinger, H.P. & Horton, W.A. (2005). Secretion of cartilage oligomeric matrix protein is affected by the signal peptide. J Biol Chem 280, 17172–17179.CrossRefGoogle ScholarPubMed
Luby-Phelps, K., Ning, G., Fogerty, J. & Besharse, J.C. (2003). Visualization of identified GFP-expressing cells by light and electron microscopy. J Histochem Cytochem 51, 271–274.CrossRefGoogle ScholarPubMed
Morozov, Y., Khalilov, I., Yehezkel, B. & Represa, A. (2002). Correlative fluorescence and electron microscopy of biocytin-filled neurons with a preservation of the postsynaptic ultrastructure. J Neurosci Methods 117, 81–85.CrossRefGoogle ScholarPubMed
Pawley, J.B. (1995). Handbook of Biological Confocal Microscopy. New York: Plenum Press.CrossRefGoogle Scholar
Pombo, A., Hollinshead, M. & Cook, P.R. (1999). Bridging and resolution gap: Imaging the same transcription factories in cryosections by light and electron microscopy. J Histochem Cytochem 47, 471–480.CrossRefGoogle ScholarPubMed
Ren, Y., Kruhlak, M.J. & Bazett-Jones, D.P. (2003). Same serial section correlative light and energy-filtered transmission electron microscopy. J Histochem Cytochem 51, 605–612.CrossRefGoogle ScholarPubMed
Sims, P.A. & Hardin, J.D. (2007). Fluorescence-integrated transmission electron microscopy images: Integrating fluorescence microscopy with transmission electron microscopy. Methods Mol Biol 369, 291–308.CrossRefGoogle ScholarPubMed