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.

  • Protocol
  • Published:

Correlative light-electron microscopy (CLEM) combining live-cell imaging and immunolabeling of ultrathin cryosections

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

Access options

Rent or buy this article

Prices vary by article type

from$1.95

to$39.95

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

Figure 1: Schematic overview of the CLEM procedure.
Figure 2: CLEM in live HepG2 cells.

References

  1. Polishchuk, R.S. et al. Correlative light-electron microscopy reveals the tubular-saccular ultrastructure of carriers operating between Golgi apparatus and plasma membrane. J. Cell. Biol. 148, 45–58 (2000).

    Article  CAS  Google Scholar 

  2. Polishchuk, E.V., Di Pentima, A., Luini, A. & Polishchuk, R.S. Mechanism of constitutive export from the golgi: bulk flow via the formation, protrusion, and en bloc cleavage of large trans-golgi network tubular domains. Mol. Biol. Cell 14, 4470–4485 (2003).

    Article  CAS  Google Scholar 

  3. Polishchuk, R.S., San Pietro, E., Di Pentima, A., Tete, S. & Bonifacino, J.S. Ultrastructure of long-range transport carriers moving from the trans Golgi network to peripheral endosomes. Traffic 7, 1092–1103 (2006).

    Article  CAS  Google Scholar 

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

  5. Grabenbauer, M. et al. Correlative microscopy and electron tomography of GFP through photooxidation. Nat. Methods 2, 857–862 (2005).

    Article  CAS  Google Scholar 

  6. Darcy, K.J., Staras, K., Collinson, L.M. & Goda, Y. An ultrastructural readout of fluorescence recovery after photobleaching using correlative light and electron microscopy. Nat. Protoc. 1, 988–994 (2006).

    Article  CAS  Google Scholar 

  7. Gaietta, G.M. et al. Golgi twins in late mitosis revealed by genetically encoded tags for live cell imaging and correlated electron microscopy. Proc. Natl. Acad. Sci. USA 103, 17777–17782 (2006).

    Article  CAS  Google Scholar 

  8. Slot, J.W. & Geuze, H.J. Cryosectioning and immunolabeling. Nat. Protoc. 2, 2480–2491 (2007).

    Article  CAS  Google Scholar 

  9. Oorschot, V., de Wit, H., Annaert, W.G. & Klumperman, J. A novel flat-embedding method to prepare ultrathin cryosections from cultured cells in their in situ orientation. J. Histochem. Cytochem. 50, 1067–1080 (2002).

    Article  CAS  Google Scholar 

  10. Falcon-Perez, J.M., Nazarian, R., Sabatti, C. & Dell'Angelica, E.C. Distribution and dynamics of Lamp1-containing endocytic organelles in fibroblasts deficient in BLOC-3. J. Cell. Sci. 118, 5243–5255 (2005).

    Article  CAS  Google Scholar 

  11. Griffith, J.M. & Posthuma, G. A reliable and convenient method to store ultrathin thawed cryosections prior to immunolabeling. 50, 57–62 (2002).

  12. van Donselaar, E., Posthuma, G., Zeuschner, D., Humbel, B.M. & Slot, J.W. Immunogold labeling of cryosections from high-pressure frozen cells. Traffic 8, 471–485 (2007).

    Article  CAS  Google Scholar 

  13. Tokuyasu, K.T. A technique for ultracryotomy of cell suspensions and tissues. J. Cell. Biol. 57, 551–565 (1973).

    Article  CAS  Google Scholar 

  14. Koster, A.J. & Klumperman, J. Electron microscopy in cell biology: integrating structure and function. Nat. Rev. Mol. Cell. Biol. 9 (Suppl.), SS6–SS10 (2003).

    Google Scholar 

  15. Griffiths, G., Pfeiffer, S., Simons, K. & Matlin, K. Exit of newly synthesized membrane proteins from the trans cisterna of the Golgi complex to the plasma membrane. J. Cell. Biol. 101, 949–964 (1985).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We thank M. v. Peski and R. Scriwanek for assistance with figure and movie preparations and all other colleagues of the Department of Cell Biology for fruitful discussions. We are grateful to R. Pepperkok (EMBL, Heidelberg) for introducing CR into live-cell imaging. We thank E. Dell'Angelica for the construct encoding LAMP-1–mGFP (University of California, Los Angeles). J.K. is the recipient of VICI grant 918.56.611 of the Netherlands Organization for Scientific research

Author information

Authors and Affiliations

Authors

Corresponding author

Correspondence to Judith Klumperman.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–2, Supplementary Table 1 (PDF 230 kb)

Supplementary Video 1

Time lapse movie showing endosomes moving with distinct dynamics. HepG2 cells were transfected with a LAMP-1-mGFP expressing construct10 and incubated with dextran and Tf-Alexa568 according to the protocol described in figure 2. LAMP-1-mGFP is present in the Golgi region and in endosomes. Note a ‘ring’ endosome that remains stationary in the Golgi region (first arrow), whereas an endosome loaded only with dextran and/or Tf-Alexa568 moves through the cytoplasm (second arrow). Still frames and CLEM images of the imaged cell are shown in figure 2. (MOV 5914 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

van Rijnsoever, C., Oorschot, V. & Klumperman, J. Correlative light-electron microscopy (CLEM) combining live-cell imaging and immunolabeling of ultrathin cryosections. Nat Methods 5, 973–980 (2008). https://doi.org/10.1038/nmeth.1263

Download citation

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nmeth.1263

This article is cited by

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