Dresden 2017 – wissenschaftliches Programm

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BP: Fachverband Biologische Physik

BP 8: Posters - Bioimaging and Spectroscopy

BP 8.13: Poster

Montag, 20. März 2017, 17:30–19:30, P3

Direct characterization of the evanescent field in objective-type total internal reflection microscopy — •Christian Niederauer, Philipp Blumhardt, Jonas Mücksch, Michael Heymann, Armin Lambacher, and Petra Schwille — Max Planck Institute of Biochemistry, Am Klopferspitz 18, 82152 Martinsried, Germany

0pt Total internal reflection fluorescence microscopy (TIRFM) is a powerful tool to study the interaction of molecules in close proximity to a surface. Usually, the axial TIRFM excitation profile is assumed to be a single-exponential with a characteristic penetration depth.

Exploiting the full potential of TIRFM data requires a precise know-ledgeknow-ledge of the excitation profile. In a first approximation, the depth of the evanescent field can be estimated from geometrical considerations. However, significant deviations from the assumed theoretical single-exponential profile have been observed in objective-type TIRFM [1].

Available methods to characterize the precise shape of the axial excitation profile require special instrumentation [2,3], sophisticated sample preparation [3] or are not applicable at typical refractive indices [1]. Here, we present our work on a new approach to quantify the axial TIRFM excitation profile. Our goal is to fabricate a robust and user-friendly micropatterned slide for in situ calibration.

[1] A.L. Mattheyses, D. Axelrod, J. of Biomed. Optics 11, 140060 (2006).

[2] Sarkar, A., et al., PNAS 101, 12882–12886 (2004).

[3] Gell, C., et al., J. of Microscopy 234, 38–46 (2009).