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J Chem Phys. 2018 Apr 7;148(13):134311. doi: 10.1063/1.5011642.

Polarized two-photon photoselection in EGFP: Theory and experiment.

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Department of Physics and Astronomy, University College London, London WC1E 6BT, United Kingdom.
Cell Biophysics Laboratory, Ikerbasque, Basque Foundation for Science and Unidad de Biofisica (CSIC-UPV/EHU), Bilbao, Spain.


In this work, we present a complete theoretical description of the excited state order created by two-photon photoselection from an isotropic ground state; this encompasses both the conventionally measured quadrupolar (K = 2) and the "hidden" degree of hexadecapolar (K = 4) transition dipole alignment, their dependence on the two-photon transition tensor and emission transition dipole moment orientation. Linearly and circularly polarized two-photon absorption (TPA) and time-resolved single- and two-photon fluorescence anisotropy measurements are used to determine the structure of the transition tensor in the deprotonated form of enhanced green fluorescent protein. For excitation wavelengths between 800 nm and 900 nm, TPA is best described by a single element, almost completely diagonal, two-dimensional (planar) transition tensor whose principal axis is collinear to that of the single-photon S0 → S1 transition moment. These observations are in accordance with assignments of the near-infrared two-photon absorption band in fluorescent proteins to a vibronically enhanced S0 → S1 transition.

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