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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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