Abstract
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.
Highlights
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
Whilst considerable effort has been directed at determining the mechanism of two-photon absorption (TPA) in fluorescent proteins,34 a full characterization of the excited states prepared by two-photon excitation has not been attempted
Our results indicate that SXX, the principal component of the two-photon transition tensor in enhanced GFP (EGFP), has the same molecular frame orientation as the single-photon S0 → S1 transition moment
Summary
The Green Fluorescent Protein (GFP) has been extensively studied and utilised in a wide range of biophysical applications following its isolation from the Aquorea victoria jellyfish. In recent years, a large number of mutants have been created to improve fluorescent yield and stability and to provide a spectrum of different absorption and emission maxima. Of these, enhanced GFP (EGFP) has become one of the most widely used tools for labelling proteins in vivo and as both a donor and an acceptor in Forster resonance energy transfer (FRET) experiments. In parallel to these developments, two-photon excitation has emerged as an important tool in fluorescence spectroscopy and microscopy, affording inherent confocal sectioning and reduced photodamage alongside the possibility of enhanced orientational photoselection.. The initial two-photon fluorescence anisotropy critically depends on both the structure of the two-photon transition tensor and the molecular frame orientation of the emission transition dipole moment, the determination of which require both linearly and circularly polarized absorption and fluorescence anisotropy measurements. Whilst considerable effort has been directed at determining the mechanism of TPA in fluorescent proteins, a full characterization of the excited states prepared by two-photon excitation has not been attempted This is of fundamental importance as, in addition to a quadrupolar (rank K = 2) degree of molecular frame and transition dipole alignment, two-photon excitation prepares the higher degree of hexadecapolar (rank K = 4) alignment. We develop the theory of polarized two-photon photoselection to determine the dependence of both K = 2 and K = 4 degrees of transition dipole alignment on the two-photon tensor structure and the molecular frame orientation of the emission transition dipole moment. A novel method (theory and experiment) for determining hexadecapolar alignment dynamics in two-photon excited states using time resolved polarized stimulated emission depletion (STED) is set out in the companion paper.
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