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Proceedings Paper

Time-resolved fluorescence microscopy
Author(s): Klaus Suhling; Carolyn Tregidgo; Nicolas Sergent; James A. Levitt; Alex Pavlides; Mark A. Green; Graham Hungerford; Ana Rei; M. Isabel C. Ferreira
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Paper Abstract

Fluorescence imaging techniques are powerful tools in the biological and biomedical sciences, because they are minimally invasive and can be applied to live cells and tissues. The fluorescence emission can be characterized not only by its intensity and position, by also by its fluorescence lifetime, polarization and wavelength. Fluorescence Lifetime Imaging (FLIM) in particular has emerged as a key technique to image the environment and interaction of specific proteins in living cells. Using a time-correlated single photon counting (TCSPC)-based FLIM set-up, we find that the fluorescence lifetime of GFP-tagged proteins in cells is a function of the refractive index of the medium the cells are suspended in. In addition, combining Fluorescence Recovery After Photobleaching (FRAP) of fluorescently labeled proteins of different sizes in sol gels with time-resolved fluorescence anisotropy measurements, we demonstrate that we can measure their lateral and rotational diffusion. This allows us to infer the size and connectivity of the pores in the sol gel matrix. Moreover, wide-field photon counting imaging, originally developed for astronomical applications, is a powerful imaging method because of its high sensitivity and excellent signal-to-noise ratio. It has a distinct advantage over CCD-based imaging due to the ability to time the arrival of individual photons. The potential of time-resolved wide-field photon counting imaging with a fast CMOS camera applied to luminescence microscopy is demonstrated.

Paper Details

Date Published: 15 October 2007
PDF: 13 pages
Proc. SPIE 6771, Advanced Photon Counting Techniques II, 677106 (15 October 2007); doi: 10.1117/12.734097
Show Author Affiliations
Klaus Suhling, King's College London (United Kingdom)
Carolyn Tregidgo, King's College London (United Kingdom)
Nicolas Sergent, King's College London (United Kingdom)
James A. Levitt, King's College London (United Kingdom)
Alex Pavlides, King's College London (United Kingdom)
Mark A. Green, King's College London (United Kingdom)
Graham Hungerford, Univ. do Minho (Portugal)
Ana Rei, Univ. do Minho (Portugal)
M. Isabel C. Ferreira, Univ. do Minho (Portugal)


Published in SPIE Proceedings Vol. 6771:
Advanced Photon Counting Techniques II
Wolfgang Becker, Editor(s)

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