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Proceedings Article

Simultaneous measurements of fluorescence lifetimes, anisotropy, and FRAP recovery curves

[+] Author Affiliations
James A. Levitt, Pei-Hua Chung, Dominic R. Alibhai, Klaus Suhling

King's College London (United Kingdom)

Proc. SPIE 7902, Imaging, Manipulation, and Analysis of Biomolecules, Cells, and Tissues IX, 79020Y (February 10, 2011); doi:10.1117/12.875151
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From Conference Volume 7902

  • Imaging, Manipulation, and Analysis of Biomolecules, Cells, and Tissues IX
  • Daniel L. Farkas; Dan V. Nicolau; Robert C. Leif
  • San Francisco, California, USA | January 22, 2011

abstract

We present fluorescence lifetime imaging (FLIM) and fluorescence anisotropy imaging along with translational diffusion measurements of living cells labelled with green fluorescent protein (GFP) recorded in a single experiment. The experimental set-up allows for time and polarization-resolved fluorescence images to be measured in every frame of a fluorescence recovery after photobleaching (FRAP) series. We have validated the method using rhodamine 123 in homogeneous solution prior to measurements of living A431 cells labelled with cdc42-GFP, for which the FRAP recovery exhibits an immobile fraction and the rotational mobility of the protein is hindered while the fluorescence lifetime fairly homogeneous across the cell. By eliminating the need for sequential measurements to extract fluorescence lifetimes and molecular diffusion coefficients we remove artefacts arising from changes in sample morphology and excessive photobleaching during sequential experiments.

© (2011) COPYRIGHT Society of Photo-Optical Instrumentation Engineers (SPIE). Downloading of the abstract is permitted for personal use only.
Citation

James A. Levitt ; Pei-Hua Chung ; Dominic R. Alibhai and Klaus Suhling
"Simultaneous measurements of fluorescence lifetimes, anisotropy, and FRAP recovery curves", Proc. SPIE 7902, Imaging, Manipulation, and Analysis of Biomolecules, Cells, and Tissues IX, 79020Y (February 10, 2011); doi:10.1117/12.875151; http://dx.doi.org/10.1117/12.875151


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