Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Article
  • Published:

Correlative microscopy and electron tomography of GFP through photooxidation

Nature Methods volume 2, pages 857–862 (2005)Cite this article

This article has been updated

Abstract

We have developed a simple correlative photooxidation method that allows for the direct ultrastructural visualization of the green fluorescent protein (GFP) upon illumination. The method, termed GRAB for GFP recognition after bleaching, uses oxygen radicals generated during the GFP bleaching process to photooxidize 3,3′-diaminobenzidine (DAB) into an electron-dense precipitate that can be visualized by routine electron microscopy and electron tomography. The amount of DAB product produced by the GRAB method appears to be linear with the initial fluorescence, and the resulting images are of sufficient quality to reveal detailed spatial information. This is exemplified by the observed intra–Golgi stack and intracisternal distribution of a human Golgi resident glycosylation enzyme, N-acetylgalactosaminyltransferase-2 fused either to enhanced GFP or CFP.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on SpringerLink
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1: Photooxidation of EGFP polymerizes DAB to an electron-dense precipitate.
Figure 2: The bleaching process and correlative light and electron microscopy of the same Golgi resident enzymes.
Figure 3: Quantitative analysis of the signal-to-noise ratio by correlative light and electron microscopy.
Figure 4: Electron tomography of a Golgi stack containing GalNAc-T2GFP.

Similar content being viewed by others

Change history

  • 28 November 2005

    Original SI Figures 1, 2, and 3 did not have figure legends in the PDF. New SI Figures 1, 2, and 3 with legends should replace the originals.

References

  1. Maranto, A.R. Neuronal mapping: a photooxidation reaction makes Lucifer yellow useful for electron microscopy. Science 217, 953–955 (1982).

    Article  CAS  Google Scholar 

  2. Gaietta, G. et al. Multicolor and electron microscopic imaging of connexin trafficking. Science 296, 503–507 (2002).

    Article  CAS  Google Scholar 

  3. Egner, A. & Hell, S.W. Fluorescence microscopy with super-resolved optical sections. Trends Cell Biol. 15, 207–215 (2005).

    Article  CAS  Google Scholar 

  4. Monosov, E.Z., Wenzel, T.J., Luers, G.H., Heyman, J.A. & Subramani, S. Labeling of peroxisomes with green fluorescent protein in living P. pastoris cells. J. Histochem. Cytochem. 44, 581–589 (1996).

    Article  CAS  Google Scholar 

  5. Svitkina, T.M. & Borisy, G.G. Correlative light and electron microscopy of the cytoskeleton of cultured cells. Methods Enzymol. 298, 570–592 (1998).

    Article  CAS  Google Scholar 

  6. Castejon, O.J. & Castejon, H.V. Correlative microscopy of cerebellar Golgi cells. Biocell 24, 13–30 (2000).

    CAS  PubMed  Google Scholar 

  7. Mironov, A.A., Polishchuk, R.S. & Luini, A. Visualizing membrane traffic in vivo by combined video fluorescence and 3D electron microscopy. Trends Cell Biol. 10, 349–353 (2000).

    Article  CAS  Google Scholar 

  8. Pagano, R.E., Sepanski, M.A. & Martin, O.C. Molecular trapping of a fluorescent ceramide analogue at the Golgi apparatus of fixed cells: interaction with endogenous lipids provides a trans-Golgi marker for both light and electron microscopy. J. Cell Biol. 109, 2067–2079 (1989).

    Article  CAS  Google Scholar 

  9. Harata, N., Ryan, T.A., Smith, S.J., Buchanan, J. & Tsien, R.W. Visualizing recycling synaptic vesicles in hippocampal neurons by FM 1–43 photoconversion. Proc. Natl. Acad. Sci. USA 98, 12748–12753 (2001).

    Article  CAS  Google Scholar 

  10. Marsh, B.J., Mastronarde, D.N., Buttle, K.F., Howell, K.E. & McIntosh, J.R. Organellar relationships in the Golgi region of the pancreatic beta cell line, HIT-T15, visualized by high resolution electron tomography. Proc. Natl. Acad. Sci. USA 98, 2399–2406 (2001).

    Article  CAS  Google Scholar 

  11. Storrie, B. et al. Recycling of golgi-resident glycosyltransferases through the ER reveals a novel pathway and provides an explanation for nocodazole-induced Golgi scattering. J. Cell Biol. 143, 1505–1521 (1998).

    Article  CAS  Google Scholar 

  12. Lubke, J. Photoconversion of diaminobenzidine with different fluorescent neuronal markers into a light and electron microscopic dense reaction product. Microsc. Res. Tech. 24, 2–14 (1993).

    Article  CAS  Google Scholar 

  13. Nilsson, T. et al. Overlapping distribution of two glycosyltransferases in the Golgi apparatus of HeLa cells. J. Cell Biol. 120, 5–13 (1993).

    Article  CAS  Google Scholar 

  14. Rabouille, C. et al. Mapping the distribution of Golgi enzymes involved in the construction of complex oligosaccharides. J. Cell Sci. 108, 1617–1627 (1995).

    CAS  PubMed  Google Scholar 

  15. Rottger, S. et al. Localization of three human polypeptide GalNAc-transferases in HeLa cells suggests initiation of O-linked glycosylation throughout the Golgi apparatus. J. Cell Sci. 111, 45–60 (1998).

    CAS  PubMed  Google Scholar 

  16. Orci, L., Perrelet, A. & Rothman, J.E. Vesicles on strings: morphological evidence for processive transport within the Golgi stack. Proc. Natl. Acad. Sci. USA 95, 2279–2283 (1998).

    Article  CAS  Google Scholar 

  17. Shorter, J., Beard, M.B., Seemann, J., Dirac-Svejstrup, A.B. & Warren, G. Sequential tethering of Golgins and catalysis of SNAREpin assembly by the vesicle-tethering protein p115. J. Cell Biol. 157, 45–62 (2002).

    Article  CAS  Google Scholar 

  18. Cosson, P., Amherdt, M., Rothman, J.E. & Orci, L. A resident Golgi protein is excluded from peri-Golgi vesicles in NRK cells. Proc. Natl. Acad. Sci. USA 99, 12831–12834 (2002).

    Article  CAS  Google Scholar 

  19. Kweon, H.S. et al. Golgi enzymes are enriched in perforated zones of golgi cisternae but are depleted in COPI vesicles. Mol. Biol. Cell 15, 4710–4724 (2004).

    Article  CAS  Google Scholar 

  20. Martinez-Menarguez, J.A. et al. Peri-Golgi vesicles contain retrograde but not anterograde proteins consistent with the cisternal progression model of intra-Golgi transport. J. Cell Biol. 155, 1213–1224 (2001).

    Article  CAS  Google Scholar 

  21. Lanoix, J. et al. GTP hydrolysis by arf-1 mediates sorting and concentration of Golgi resident enzymes into functional COP I vesicles. EMBO J. 18, 4935–4948 (1999).

    Article  CAS  Google Scholar 

  22. Lin, C.C., Love, H.D., Gushue, J.N., Bergeron, J.J. & Ostermann, J.E.R. Golgi intermediates acquire Golgi enzymes by brefeldin A–sensitive retrograde transport in vitro. J. Cell Biol. 147, 1457–1472 (1999).

    Article  CAS  Google Scholar 

  23. Malsam, J., Satoh, A., Pelletier, L. & Warren, G. Golgin tethers define subpopulations of COPI vesicles. Science 307, 1095–1098 (2005).

    Article  CAS  Google Scholar 

  24. McIntosh, R., Nicastro, D. & Mastronarde, D. New views of cells in 3D: an introduction to electron tomography. Trends Cell Biol. 15, 43–51 (2005).

    Article  CAS  Google Scholar 

  25. Paabo, S., Weber, F., Nilsson, T., Schaffner, W. & Peterson, P.A. Structural and functional dissection of an MHC class I antigen-binding adenovirus glycoprotein. EMBO J. 5, 1921–1927 (1986).

    Article  CAS  Google Scholar 

  26. Baschong, W., Suetterlin, R. & Laeng, R.H. Control of autofluorescence of archival formaldehyde-fixed, paraffin-embedded tissue in confocal laser scanning microscopy (CLSM). J. Histochem. Cytochem. 49, 1565–1572 (2001).

    Article  CAS  Google Scholar 

  27. Hayat, M.A. Principles and techniques of electron microscopy. (Van Nostrand Reinhold Company, New York, 1970).

  28. Ziese, U. et al. Automated high-throughput electron tomography by pre-calibration of image shifts. J. Microsc. 205, 187–200 (2002).

    Article  CAS  Google Scholar 

  29. Kremer, J.R., Mastronarde, D.N. & McIntosh, J.R. Computer visualization of three-dimensional image data using IMOD. J. Struct. Biol. 116, 71–76 (1996).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We wish to thank R. Pepperkok, J. Rietdorf and K. Miura (Advanced Light Microscopy Facility, EMBL Heidelberg) for discussions and help in image analysis, M. Lebbink (Utrecht, The Netherlands) for his help using Amira software, M. Axelsson for help in preparing mitotic populations of HeLa cells and Swegene for its support of the Center for Cellular Imaging in Gothenburg. HeLa cells stably expressing α-tubulin-EGFP were a generous gift of J. Lipp and J.-M. Peters (IMP Vienna, Austria). This work was supported by an EMBO fellowship (M.G.).

Author information

Authors and Affiliations

  1. Cell Biology and Cell Biophysics Program, EMBL, Meyerhofstr. 1, Heidelberg, D-69117, Germany

    Markus Grabenbauer & Andreas Hoenger

  2. Department of Molecular Cell Biology, Institute of Biomembranes, Utrecht University, Utrecht, 3584 CH, The Netherlands

    Willie J C Geerts & Abraham J Koster

  3. Department of Medical Biochemistry and Swegene Center for Cellular Imaging, Göteborg University, Medicinaregatan 9A, Göteborg, 413 90, Sweden

    Julia Fernadez-Rodriguez & Tommy Nilsson

Authors
  1. Markus Grabenbauer
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Willie J C Geerts
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Julia Fernadez-Rodriguez
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Andreas Hoenger
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Abraham J Koster
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Tommy Nilsson
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Tommy Nilsson.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Fig. 1

HeLa cells stably expressing GalNAc-T2CFP were cultivated for 22 hours in 100μM nocodazole, then fixed and processed for photooxidation. (PDF 771 kb)

Supplementary Fig. 2

HeLa cells were infected with a recombinant adenovirus strain expressing the temperature sensitive vesicular stomatitis virus (VSV) G protein derived from the VSV Orsay ts045 strain and fused to ECFP. (PDF 1417 kb)

Supplementary Fig. 3

Peri-Golgi vesicles in perpendicular projections. (PDF 1487 kb)

Supplementary Fig. 4

Microtubules. (PDF 1093 kb)

Supplementary Video 1

Tomography study of a part of the Golgi stack containing DAB-precipitate representing the Golgi-resident enzyme, GalNAc-T2EGFP. It starts with a ‘virtual flight through’ of the tomogram in z-axis direction. This is followed by the manual tracing of membranes to yield a 3D representation of the Golgi stack in different colors for each cisterna. Rotation of the final 3D representation shows GFP-containing cisternae in green with a highlight of the peri-Golgi vesicle found to contain DAB-precipitate. (MOV 2479 kb)

Supplementary Video 2

A detailed 3D representation of the peri-Golgi vesicle found to contain DAB-precipitate and adjacent cisternae. Note the small protrusions that extend away from the cisternae towards the vesicle. (MOV 1391 kb)

Supplementary Methods (PDF 94 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Grabenbauer, M., Geerts, W., Fernadez-Rodriguez, J. et al. Correlative microscopy and electron tomography of GFP through photooxidation. Nat Methods 2, 857–862 (2005). https://doi.org/10.1038/nmeth806

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nmeth806

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing