Time-resolved fluorescence microscopy

Klaus Suhling; Carolyn Tregidgo; Nicolas Sergent; James A. Levitt; Alex Pavlides; Mark A. Green; Graham Hungerford; Ana Rei; M. Isabel C. Ferreira

doi:10.1117/12.734097

15 October 2007 Time-resolved fluorescence microscopy

Klaus Suhling, Carolyn Tregidgo, Nicolas Sergent, James A. Levitt, Alex Pavlides, Mark A. Green, Graham Hungerford, Ana Rei, M. Isabel C. Ferreira

Author Affiliations +

Proceedings Volume 6771, Advanced Photon Counting Techniques II; 677106 (2007) https://doi.org/10.1117/12.734097
Event: Optics East, 2007, Boston, MA, United States

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.

Citation Download Citation

Klaus Suhling, Carolyn Tregidgo, Nicolas Sergent, James A. Levitt, Alex Pavlides, Mark A. Green, Graham Hungerford, Ana Rei, and M. Isabel C. Ferreira "Time-resolved fluorescence microscopy", Proc. SPIE 6771, Advanced Photon Counting Techniques II, 677106 (15 October 2007); https://doi.org/10.1117/12.734097

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