Time resolved 3D orientation spectroscopy: experimental realization and simulation

Richard Börner; Danny Kowerko; Christian von Borczyskowski; Christian G. Hübner

doi:10.1117/12.874897

11 February 2011 Time resolved 3D orientation spectroscopy: experimental realization and simulation

Richard Börner, Danny Kowerko, Christian von Borczyskowski, Christian G. Hübner

Proceedings Volume 7905, Single Molecule Spectroscopy and Imaging IV; 79050D (2011) https://doi.org/10.1117/12.874897
Event: SPIE BiOS, 2011, San Francisco, California, United States

Abstract

Confocal microscopy is a powerful tool for single molecule investigation of fluorescent macromolecules. Besides the commonly studied features in single molecule detection, the 3D orientation determination of the emission dipole enables the analysis of different conformational states. These conformational states can be represented as state depending dipole orientations intrinsic to the fluorescent molecule and/or in relation to the molecular frame. They might be subject to intramolecular dynamics, which may lead to spectral diffusion, fluorescence intensity and/or lifetime fluctuations and changes in the orientation of the emission dipole. We demonstrate a detection scheme that allows for simultaneous determination of the full 3D emission dipole orientation, the fluorescence intensity, the fluorescence lifetime and the emission spectra of single fluorescent molecules. We evaluate the feasibility of our approach using pyridyl functionalized perylene bisimide (PBI) as a model system exhibiting conformational changes. Moreover, MC simulations demonstrate the full potential of our detection scheme to distinguish between intensity fluctuations due to conformational changes and changes in the out-of-plane orientation or changes in both of them.

Citation Download Citation

Richard Börner, Danny Kowerko, Christian von Borczyskowski, and Christian G. Hübner "Time resolved 3D orientation spectroscopy: experimental realization and simulation", Proc. SPIE 7905, Single Molecule Spectroscopy and Imaging IV, 79050D (11 February 2011); https://doi.org/10.1117/12.874897

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