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Correlative microscopy and electron tomography of GFP through photooxidation

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

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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.

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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.

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  • 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.

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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.).

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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
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  2. Willie J C Geerts
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  4. Andreas Hoenger
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  5. Abraham J Koster
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  6. Tommy Nilsson
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Correspondence to Tommy Nilsson.

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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)

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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

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