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BP: Fachverband Biologische Physik

BP 15: Single-Molecule Biophysics I

BP 15.6: Talk

Tuesday, March 15, 2011, 12:00–12:15, ZEU 250

Scanning evanescent fields in TIRF microscopy using a single point-like light source and a DNA worm drive — •Hergen Brutzer, Friedrich W. Schwarz, and Ralf Seidel — Biotechnologisches Zentrum, TU Dresden, Tatzberg 47/49, 01307 Dresden

Total internal reflection fluorescence (TIRF) microscopy is an elegant technique that limits the dimension of the excitation volume along the z-direction to the hundred nanometer-scale. The method makes use of the evanescent field arising when light is totally internally reflected at the boundary to a medium of lower refractive index. Often the penetration depth of this exponentially decaying field is left undetermined limiting the reproducibility in different experiments. We directly measure this quantity by using a quantum dot as a point-like light source and a Holliday junction as a drive to move the fluorescent probe with nanometer precision along the z-direction. The junction serves as a worm drive, which couples rotation into translational movement, while the DNA pitch serves as an intrinsic ruler. The junction is forced to migrate by adding negative turns to the DNA stretched perpendicular to the surface using magnetic tweezers. This causes the quantum dot, which is attached upstream of the junction, to decrease its height above the surface by 3.4 nm per turn. Thus it can be moved continuously through the excitation field while monitoring its height-dependent fluorescent signal. Since the quantum dot is a point-like light source, the intensity decay of the evanescent field can be obtained by dividing the signal recorded in TIRF illumination by the one recorded in conventional epi-illumination without further corrections.