Protocol

Cytoskeleton Methods and Protocols

Volume 586 of the series Methods in Molecular Biology pp 117-142

Date:

Quantitative Fluorescence Microscopy Techniques

  • Alessandro EspositoAffiliated withDepartment of Chemical Engineering and Biotechnology, University of Cambridge
  • , Simon SchlachterAffiliated withDepartment of Chemical Engineering and Biotechnology, University of Cambridge
  • , Gabriele S. Kaminski SchierleAffiliated withDepartment of Chemical Engineering and Biotechnology, University of Cambridge
  • , Alan D. ElderAffiliated withDepartment of Chemical Engineering and Biotechnology, University of Cambridge
  • , Alberto DiasproAffiliated withLAMBS-MicroScoBio, Department of Physics, University of Genoa
  • , Fred S. WoutersAffiliated withLaboratory for Molecular and Cellular Systems, Department of Neuro- and Sensory Physiology, University Medicine Göttingen
  • , Clemens F. KaminskiAffiliated withDepartment of Chemical Engineering and Biotechnology, University of Cambridge
  • , Asparouh I. IlievAffiliated withRudolf Virchow Center & Institute of Pharmacology and Toxicology, University of Würzburg

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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.

Key words

FRET FLIM FRAP FLAP FLIP Bioimaging