Berlin 2008 – wissenschaftliches Programm

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BP: Fachverband Biologische Physik

BP 15: Single Molecules

BP 15.10: Vortrag

Mittwoch, 27. Februar 2008, 16:30–16:45, C 243

TIRFM evanescent field calibration using tilted microtubules — •Chris Gell, Michael Berndt, and Stefan Diez — MPI-CBG, Dresden, Germany

Total internal reflection fluorescence microscopy (TIRFM) has become a powerful tool to study the dynamics of subcellular structures and single molecules near substrate surfaces. However, the penetration depth of the evanescent field , i.e. the distance at which the excitation intensity has exponentially decayed to 1/e, is often left undetermined. This presents a limit on the spatial information about the imaged structures. Moreover, in multi-color TIRFM applications, e.g. to perform colocalization studies, it is crucial to ensure equal penetration depths for the different excitation wavelengths. Here, we present a novel method to quantitatively characterise the illumination in TIRFM using tilted, fluorescently labelled, microtubules. Importantly, the use of in vitro reconstituted microtubules as nanoscale rulers results in a minimal perturbation of the evanescent field. Excitation light scattering is essentially eliminated and the refractive index of the sample environment is virtually unchanged. Our method has the potential to provide a generic tool for in-situ calibration of the evanescent field.