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Identifying Dynamic Membrane Structures with Atomic-Force Microscopy and Confocal Imaging

Published online by Cambridge University Press:  13 February 2014

Tobias Timmel ,
Markus Schuelke  and
Simone Spuler
Tobias Timmel*
Affiliation:
Muscle Research Unit, Experimental and Clinical Research Center, a joint cooperation between the Charité Medical Faculty and the Max Delbrück Center for Molecular Medicine Berlin, Lindenberger Weg 80, D-13125 Berlin, Germany
Markus Schuelke
Affiliation:
Department of Neuropediatrics and NeuroCure Clinical Research Center, Charité Universitätsmedizin, Augustenburger Platz 1, D-13353Berlin, Germany
Simone Spuler
Affiliation:
Muscle Research Unit, Experimental and Clinical Research Center, a joint cooperation between the Charité Medical Faculty and the Max Delbrück Center for Molecular Medicine Berlin, Lindenberger Weg 80, D-13125 Berlin, Germany
*
*Corresponding author. tobias.timmel@charite.de
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Abstract

Combining the biological specificity of fluorescence microscopy with topographical features revealed by atomic force microscopy (AFM) provides new insights into cell biology. However, the lack of systematic alignment capabilities especially in scanning-tip AFM has limited the combined application approach as AFM drift leads to increasing image mismatch over time. We present an alignment correction method using the cantilever tip as a reference landmark. Since the precise tip position is known in both the fluorescence and AFM images, exact re-alignment becomes possible. We used beads to demonstrate the validity of the method in a complex artificial sample. We then extended this method to biological samples to depict membrane structures in fixed and living human fibroblasts. We were able to map nanoscale membrane structures, such as clathrin-coated pits, to their respective fluorescent spots. Reliable alignment between fluorescence signals and topographic structures opens possibilities to assess key biological processes at the cell surface such as endocytosis and exocytosis.

Keywords

atomic force microscopyconfocal microscopyfluorescence imagingimage alignmentcaveolaeclathrin-coated pits
Type
Biological Applications
Information
Microscopy and Microanalysis , Volume 20 , Issue 2 , April 2014 , pp. 514 - 520
Copyright
© Microscopy Society of America 2014 

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References

Barattin, R. & Voyer, N. (2011). Chemical modifications of atomic force microscopy tips. Methods Mol Biol 736, 457–483.CrossRefGoogle ScholarPubMed
Bastiani, M. & Parton, R.G. (2010). Caveolae at a glance. J Cell Sci 123(Pt 22), 3831–3836.CrossRefGoogle ScholarPubMed
Bretscher, M.S., Thomson, J.N. & Pearse, B.M. (1980). Coated pits act as molecular filters. Proc Natl Acad Sci USA 77(7), 4156–4159.Google Scholar
Defranchi, E., Bonaccurso, E., Tedesco, M., Canato, M., Pavan, E., Raiteri, R. & Reggiani, C. (2005). Imaging and elasticity measurements of the sarcolemma of fully differentiated skeletal muscle fibres. Microsc Res Tech 67(1), 27–35.Google Scholar
Flores, S.M. & Toca-Herrera, J.L. (2009). The new future of scanning probe microscopy: Combining atomic force microscopy with other surface-sensitive techniques, optical microscopy and fluorescence techniques. Nanoscale 1(1), 40–49.CrossRefGoogle Scholar
Frankel, D.J., Pfeiffer, J.R., Surviladze, Z., Johnson, A.E., Oliver, J.M., Wilson, B.S. & Burns, A.R. (2006). Revealing the topography of cellular membrane domains by combined atomic force microscopy/fluorescence imaging. Biophys J 90(7), 2404–2413.Google Scholar
Gaiduk, A., Kühnemuth, R., Antonik, M. & Seidel, C.A.M. (2005). Optical characteristics of atomic force microscopy tips for single-molecule fluorescence applications. Chemphyschem 6(5), 976–983.CrossRefGoogle ScholarPubMed
Hill, M.M., Bastiani, M., Luetterforst, R., Kirkham, M., Kirkham, A., Nixon, S.J., Walser, P., Abankwa, D., Oorschot, V.M.J., Martin, S., Hancock, J.F. & Parton, R.G. (2008). PTRF-Cavin, a conserved cytoplasmic protein required for caveola formation and function. Cell 132(1), 113–124.CrossRefGoogle ScholarPubMed
Hutter, J.L. & Bechhoefer, J. (1993). Calibration of atomic-force microscope tips. Rev Sci Instrum 64(7), 1868–1873.Google Scholar
Kassies, R., van der Werf, K.O., Lenferink, A., Hunter, C.N., Olsen, J.D., Subramaniam, V. & Otto, C. (2005). Combined AFM and confocal fluorescence microscope for applications in bio-nanotechnology. J Microsc 217(Pt 1), 109–116.CrossRefGoogle ScholarPubMed
Kellermayer, M.S.Z. (2011). Combined atomic force microscopy and fluorescence microscopy. Methods Mol Biol 736, 439–456.Google Scholar
Kienberger, F., Pastushenko, V.P., Kada, G., Puntheeranurak, T., Chtcheglova, L., Riethmueller, C., Rankl, C., Ebner, A. & Hinterdorfer, P. (2006). Improving the contrast of topographical AFM images by a simple averaging filter. Ultramicroscopy 106(8–9), 822–828.Google Scholar
Loerke, D., Mettlen, M., Yarar, D., Jaqaman, K., Jaqaman, H., Danuser, G. & Schmid, S.L. (2009). Cargo and dynamin regulate clathrin-coated pit maturation. PLoS Biol 7(3), e57.CrossRefGoogle ScholarPubMed
Lucius, H., Friedrichson, T., Kurzchalia, T.V. & Lewin, G.R. (2003). Identification of caveolae-like structures on the surface of intact cells using scanning force microscopy. J Membr Biol 194(2), 97–108.Google Scholar
Meijering, E., Dzyubachyk, O. & Smal, I. (2012). Methods for cell and particle tracking. Methods Enzymol 504, 183–200.CrossRefGoogle ScholarPubMed
Müller, D.J., Schabert, F.A., Büldt, G. & Engel, A. (1995). Imaging purple membranes in aqueous solutions at sub-nanometer resolution by atomic force microscopy. Biophys J 68(5), 1681–1686.Google Scholar
Necas, D. & Klapetek, P. (2012). Gwyddion: An open-source software for SPM data analysis. Cent Eu J Phys 10, 181–188.Google Scholar
Rajab, A., Straub, V., McCann, L.J., Seelow, D., Varon, R., Barresi, R., Schulze, A., Lucke, B., Lützkendorf, S., Karbasiyan, M., Bachmann, S., Spuler, S. & Schuelke, M. (2010). Fatal cardiac arrhythmia and long-QT syndrome in a new form of congenital generalized lipodystrophy with muscle rippling (CGL4) due to PTRF-CAVIN mutations. PLoS Genet 6(3), e1000874.Google Scholar
Schindelin, J., Arganda-Carreras, I., Frise, E., Kaynig, V., Longair, M., Pietzsch, T., Preibisch, S., Rueden, C., Saalfeld, S., Schmid, B., Tinevez, J.-Y., White, D.J., Hartenstein, V., Eliceiri, K., Tomancak, P. & Cardona, A. (2012). Fiji: An open-source platform for biological-image analysis. Nat Methods 9(7), 676–682.Google Scholar
Shevchuk, A.I., Novak, P., Taylor, M., Diakonov, I.A., Ziyadeh-Isleem, A., Bitoun, M., Guicheney, P., Lab, M.J., Gorelik, J., Merrifield, C.J., Klenerman, D. & Korchev, Y.E. (2012). An alternative mechanism of clathrin-coated pit closure revealed by ion conductance microscopy. J Cell Biol 197(4), 499–508.Google Scholar
Wu, X., Zhao, X., Baylor, L., Kaushal, S., Eisenberg, E. & Greene, L.E. (2001). Clathrin exchange during clathrin-mediated endocytosis. J Cell Biol 155(2), 291–300.CrossRefGoogle ScholarPubMed

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