On the measurement of molecular anisotropies using laser techniques. I. Polarized laser fluorescence

The tensor density matrix formalism is used to derive expressions for the circular and linear polarization of laser‐induced fluorescence from molecules which have an anisotropic distribution in the spatial orientation of their ground state angular momentum components. The generalized anisotropic distribution is expressed as a series of state multipolar moments and it is shown that the excited state multipolar moments created therefrom by the absorption of laser radiation may be quite complex even in the absence of perturbations which cause cross relaxation. Under these circumstances, polarized laser fluorescence does not give an unambiguous measure of the ground state multipolar moments and in succeeding papers we discuss methods which do yield these quantities without ambiguity

On the measurement of molecular anisotropies using laser techniques. III. Detection of the higher multipoles

In this paper we discuss the problem of measuring the higher moments (K≥2) of a generalized anisotropic distribution of molecular rotors. Two photon absorption techniques may be used to obtain these quantities and the appropriate expressions for linear and circular dichroism are derived

Complete determination of the state multipoles of rotationally resolved polarized fluorescence using a single experimental geometry

When laser radiation is used to prepare single rovibronic levels in molecules, the excited state Mj distribution is invariably polarized. In many such experiments the polarization of the excited state is ignored, which is an inadequate basis for accurate work as much valuable detail is lost. A better approach is a completely polarization resolved experiment in which the preparation, dynamics, and detection of the excited state polarization components (the state multipoles JJρQ K) are fully described. A treatment of polarized excitation in terms of the state multipoles JJρQ K is presented and consideration of excited state symmetry indicates that a common experimental geometry for linearly and circularly polarized excitation is feasible. A complete determination of the state multipoles (K=0,1,2) is shown to be possible within a single experimental geometry. It is shown that neglect of polarization phenomena can lead to ambiguities in the interpretation of some experiments

On the measurement of molecular anisotropies using laser techniques. II. Differential absorption of circularly and linearly polarized light

In this paper we describe a method which yields an unambiguous measure of the state multipolar moments of an anisotropic array of the angular momentum components of an assembly of ground state molecules. The method involves the measurement of circularly and linearly dichroic, single photon absorption and through such measurements, state moments having AT≤2 may be directly obtained. The advantages of this technique over polarized laser fluorescence are discussed

Time-resolved stimulated emission depletion and energy transfer dynamics in two-photon excited EGFP

Time and polarization-resolved stimulated emission depletion (STED) measurements are used to investigate excited state evolution following the two-photon excitation of enhanced green fluorescent protein (EGFP). We employ a new approach for the accurate STED measurement of the hitherto unmeasured degree of hexadecapolar transition dipole moment alignment ⟨α40⟩ present at a given excitation-depletion (pump-dump) pulse separation. Time-resolved polarized fluorescence measurements as a function of pump-dump delay reveal the time evolution of ⟨α40⟩ to be considerably more rapid than predicted for isotropic rotational diffusion in EGFP. Additional depolarization by homo-Förster resonance energy transfer is investigated for both ⟨α20⟩ (quadrupolar) and ⟨α40⟩ transition dipole alignments. These results point to the utility of higher order dipole correlation measurements in the investigation of resonance energy transfer processes

Formation of a long‐lived collision complex in alkali diatomic–rare gas collisions and rotational excitation via a mode‐specific dissociation

We report an unusual pressure dependence of the gas phase dynamics of alkali diatomics in 1Π excited states in their interactions with certain collision partners. This behavior consists of a change in the relative rates of the ΔJ=+1 and ΔJ=−1 collision‐induced transitions, which results in a dominance of rotational excitation at high pressures. This effect occurs only for the e Λ‐doublet component of the excited 1Π state. Also observed is a change in the polarization of the parent line such that the polarization ratio increases with increasing pressure. The dependence of this behavior on intermolecular potential, reduced duration of collision, rotational state, and temperature lead us to propose that a long‐lived complex or orbiting resonance is responsible for the effects observed. This species appears to be formed during the lifetime of the excited state of the diatomic. Knowledge of the molecule‐fixed geometry of the Λ doublets allows us to propose a structure for the long‐lived species and to speculate that the observed characteristic rotational excitation results from specific vibrational and rotational predissociation processes within the complex

Polarized two-photon photoselection in EGFP: Theory and experiment

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

Heterogeneity and restricted state selection in FRET with fluorescent proteins

Most fluorescent proteins exhibit multi-exponential fluorescence decays, indicating the presence of a heterogeneous excited state population. In the analysis of FRET to and between fluorescent proteins, it is often convenient to assume that a single interaction pathway is involved. However, in recent work we have shown that this assumption does not hold. Moreover, certain pathways can be highly constrained, leading to the potential misinterpretation of experimental data concerning protein-protein interactions. FRET and single-photon absorption both obey the same global electric dipole selection rules but differ greatly in the mechanism of the acceptor photoselection. In an isotropic medium, single-photon excitation accesses all acceptor transition dipole moment orientations with an equal probability. However, the FRET rate depends on the relative orientation of the donor and acceptor through the κ2 orientation parameter. We show how time- and spectrally- resolved fluorescence intensity and anisotropy decay measurements following direct acceptor excitation, combined with those of the interacting FRET pair, can be used to identify restricted FRET state selection and thus provide accurate measurements of protein-protein interaction dynamics