Berlin 2008 – scientific programme

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BP: Fachverband Biologische Physik

BP 7: Posters I

BP 7.13: Poster

Monday, February 25, 2008, 17:00–19:30, Poster A

Three-dimensional Fluorescence Lifetime Imaging with a light sheet based microscope (SPIM-FLIM) provides an excellent signal-to-noise ratio — •Manuel J. Neetz, Klaus Greger, Emmanuel G. Reynaud, and Ernst H.K. Stelzer — European Molecular Biology Laboratory (EMBL), Meyerhofstrasse 1, D-69117 Heidelberg, Germany

Fluorescence Lifetime Imaging (FLIM) (Gadella, 1993) in combination with modern microscopes allows one to determine the spatial distribution of specific molecular interactions. A FLIM setup operating on three dimensional (3D) live specimens requires optical sectioning capabilities, a low sample exposure and a physiologically relevant environment (Pampaloni, 2007). The optical sectioning capabilities of scanning approaches are limited due to high photobleaching rates and long acquisition times while widefield based setups do not provide optical sectioning at a reasonable noise level. The combination of EMBL's Single Plane Illumination Microscopy (SPIM) (Huisken, 2004; Greger, 2007) with frequency domain FLIM overcomes these limitations by decreasing photobleaching rates as well as increasing the signal-to-noise ratio significantly. It thus utilizes the fluorophores more efficiently. Hence, our approach is particularly well suited for investigating cellular interactions in 3D. We use our setup to acquire fluorescence lifetimes of EGFP labelled E-Cadherin in complex multi-cellular MDCK cysts to study cell-cell adhesion under relevant conditions. E.g., the ratio between cell surfaces in contact with each other and their volumes is manyfold as compared to conventional assays.