CLEO/EUROPE ; EQEC European Quantum Electronics Conference, Munich ICM, Germany, 22-27 June, 2003N

Davis, D.M.

French, Paul M.W.

Lanigan, P.

Lévêque-Fort, S.

Phillips, D.

Sabharwal, Y.

Siegel, Jan

Suhling, K.

Webb, S.E.D.

Digital.CSIC

(a) (b)Fig 1: a) A FLIM image of a B cell stained with thefluorescein derivative CFSE. b) The correspondingrotational correlation time image. The images whereacquired simultaneously in 75 seconds.Mapping the rotational diffusion of fluorophores in cells with time-resolvedwide-field fluorescence anisotropy imagingK. Suhling 1,2,3, J. Siegel 1,5, S. Lévêque-Fort 1,6, S.E.D. Webb 1, P. Lanigan 1, Y. Sabharwal 4, D. Phillips 3, D.M. Davis 2and P.M.W. French 11Photonics Group, Department of Physics, 2Department of Biological Sciences, 3Department of Chemistry, ImperialCollege of Science, Technology & Medicine, London SW7 2BW, UK, tel +44 20 7594 7715, fax +44 20 7594 77144Optical Insights, LLC, 1807 2nd  Street, Suite 1, Santa Fe, NM 87505, USA5present address: Instituto de Optica (CSIC), 28006 Madrid, Spain6present address: Laboratoire de Photophysique Moleculaire, 91405 Orsay, FranceAbstract:We demonstrate time-resolved fluorescence anisotropy imaging with simultaneous acquisition of the paralleland perpendicular polarization of a fluorescence image. This allows imaging of the viscosity in cells.SummaryFor analysis of sample arrays in assays and for imaging of molecular biology, it is desirable to develop techniques forcontrasting both different biological species and also the local environment of such species. Fluorescent labels provide ameans not only to visualise the distribution of target species but also to probe their local environment through thefluorescence lifetime. This approach is attractive since it is based on relative intensity measurements and is independentof fluorophore concentration. Here, we report the extension of picosecond time-gated fluorescence lifetime imaging(FLIM) to time-resolved fluorescence anisotropy imaging (TR-FAIM).1 Upon excitation with linearly polarized laserpulses, the parallel and perpendicular components of the fluorescence emission are imaged simultaneously using aPolarization-Resolved Imager. This yields both the rotational correlation time and fluorescence lifetime in each pixel ofthe image, the former providing information about therotational diffusion of a fluorophore.  To demonstrate thetechnique, we imaged B cells using the fluoresceinderivative CFSE (carboxyfluorescein diacetate succinimidylester), which stains the cytoplasm. The FLIM image showsa homogeneous distribution of the fluorophore and has anaverage fluorescence lifetime of 3.46±0.16ns (Fig 1a)). Therotational correlation time reports on the viscosity of thecytoplasm, and the corresponding preliminary image isshown in Fig 1b). The average value for the rotationalcorrelation time is 4.50±0.87ns at 20˚C, which correspondsto an average cytoplasmic viscosity of 14cp. This is in goodagreement with 10-13cp at 23˚C quoted previously.2  Thisillustrates the potential to image, for example, viscosityvariations in live cells. Moreover, as the rotational diffusioncan be slowed down by binding as well as otherconformational changes, the technique has potential tovisualise, for example, the binding of ligands and receptorsin cell biology.References[1] Wide-field time-resolved fluorescence anisotropy imaging (TR-FAIM) – Imaging the rotational mobility of afluorophore. J. Siegel, K. Suhling, S. Lévêque-Fort, S.E.D. Webb, D.M. Davis, D. Phillips, Y. Sabharwal and P.M.W.French. Review of Scientific Instruments, in press (Jan 2003).[2] Mapping of fluorescence anisotropy in living cells by ratio imaging. Applications to cytoplasmic viscosity.J.A. Dix and A.S. Verkman. Biophysical Journal, 57, 231-240, 1990.

Mapping the rotational diffusion of fluorophores in cells with time-resolved  wide-field fluorescence anisotropy imaging

Mapping the rotational diffusion of fluorophores in cells with time-resolved wide-field fluorescence anisotropy imaging

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