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Cytoskeleton Methods and Protocols pp 117–142Cite as

Quantitative Fluorescence Microscopy Techniques

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Part of the book series: Methods in Molecular Biology ((MIMB,volume 586))

Summary

Fluorescence microscopy is a non-invasive technique that allows high resolution imaging of cytoskeletal structures. Advances in the field of fluorescent labelling (e.g., fluorescent proteins, quantum dots, tetracystein domains) and optics (e.g., super-resolution techniques and quantitative methods) not only provide better images of the cytoskeleton, but also offer an opportunity to quantify the complex of molecular events that populate this highly organised, yet dynamic, structure.

For instance, fluorescence lifetime imaging microscopy and Förster resonance energy transfer imaging allow mapping of protein–protein interactions; furthermore, techniques based on the measurement of photobleaching kinetics (e.g., fluorescence recovery after photobleaching, fluorescence loss in photobleaching, and fluorescence localisation after photobleaching) permit the characterisation of axonal transport and, more generally, diffusion of relevant biomolecules.

Quantitative fluorescence microscopy techniques offer powerful tools for understanding the physiological and pathological roles of molecular machineries in the living cell.

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Acknowledgements

AE is funded by the Engineering and Physical Sciences Research Council (EPSRC, EP/F044011/1), UK. AII is financed by the Emmy Noether Programme of the German Science Foundation (Deutsche Forschungsgemeinschaft (DFG)) and the DFG-funded Rudolf Virchow Zentrum for Experimental Medicine at the University of Würzburg, Germany. CFK is funded by the Leverhulme Trust (UK), the Biotechnology and Biological Sciences Research Council (BBSRC, UK) and the EPSRC. FSW is financed by the Excellence Cluster 171 “Microscopy on the Nanometer Scale” of the DFG-funded Center for Molecular Physiology of the Brain (CMPB) and by the German Federal Ministry for Education and Research (BMBF). AD is funded by the University of Genoa and the Ministry of Education, University and Research (MIUR).

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Authors and Affiliations

  1. Department of Chemical Engineering and Biotechnology, University of Cambridge, Cambridge, UK

    Alessandro Esposito, Simon Schlachter, Gabriele S. Kaminski Schierle, Alan D. Elder & Clemens F. Kaminski

  2. LAMBS-MicroScoBio, Department of Physics, University of Genoa, Genoa, Italy

    Alberto Diaspro

  3. Laboratory for Molecular and Cellular Systems, Department of Neuro- and Sensory Physiology, University Medicine Göttingen, Göttingen, Germany

    Fred S. Wouters

  4. Rudolf Virchow Center & Institute of Pharmacology and Toxicology, University of Würzburg, Würzburg, Germany

    Asparouh I. Iliev

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  1. Alessandro Esposito
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  1. Brooklyn College, City University of New York, Bedford Ave. 2900, New York, 11210-2889, U.S.A.

    Ray H. Gavin

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Esposito, A. et al. (2009). Quantitative Fluorescence Microscopy Techniques. In: Gavin, R. (eds) Cytoskeleton Methods and Protocols. Methods in Molecular Biology, vol 586. Humana Press. https://doi.org/10.1007/978-1-60761-376-3_6

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