Conformation of self-assembled porphyrin dimers in liposome vesicles by phase-modulation 2D fluorescence spectroscopy.

By applying a phase-modulation fluorescence approach to 2D electronic spectroscopy, we studied the conformation-dependent exciton coupling of a porphyrin dimer embedded in a phospholipid bilayer membrane. Our measurements specify the relative angle and separation between interacting electronic transition dipole moments and thus provide a detailed characterization of dimer conformation. Phase-modulation 2D fluorescence spectroscopy (PM-2D FS) produces 2D spectra with distinct optical features, similar to those obtained using 2D photon-echo spectroscopy. Specifically, we studied magnesium meso tetraphenylporphyrin dimers, which form in the amphiphilic regions of 1,2-distearoyl-sn-glycero-3-phosphocholine liposomes. Comparison between experimental and simulated spectra show that although a wide range of dimer conformations can be inferred by either the linear absorption spectrum or the 2D spectrum alone, consideration of both types of spectra constrain the possible structures to a "T-shaped" geometry. These experiments establish the PM-2D FS method as an effective approach to elucidate chromophore dimer conformation.

II. Kinetic pathways of switching optical conformations in DsRed by 2D Fourier imaging correlation spectroscopy.

The kinetics of biomolecular conformational transitions can be studied by two-dimensional (2D) magnetic resonance and optical spectroscopic methods. Here we apply polarization-modulated Fourier imaging correlation spectroscopy (PM-FICS) to demonstrate a new approach to 2D optical spectroscopy. PM-FICS enables measurements of conformational fluctuations of fluorescently labeled macromolecules on a broad range of time scales (10(-3)-10(2) s). We examine the optical switching pathways of DsRed, a tetrameric complex of fluorescent protein subunits. An analysis of PM-FICS coordinate trajectories, in terms of 2D spectra and joint probability distributions, provides detailed information about the transition pathways between distinct dipole-coupled DsRed conformations.

I. Conformational dynamics of biological macromolecules by polarization-modulated Fourier imaging correlation spectroscopy.

Experiments that optically probe the translational motions and internal conformational transitions of biological macromolecules have the potential to enable mechanistic studies of biochemical processes in living cells. This work presents a novel "phase-selective" approach to fluorescence fluctuation spectroscopy that simultaneously monitors protein conformational transitions and nanometer center-of-mass displacements. Polarization- and intensity-modulated photoexcitation is combined with phase-sensitive signal detection to monitor the collective coordinate fluctuations from a large population of fluorescent molecules (N approximately 10(6)). Test experiments are performed on DsRed, a tetrameric complex of fluorescent protein subunits. Thermally induced conformational transitions of the complex lead to fluctuations in the optical dipolar coupling between adjacent chromophore sites. Polarization-resolved equilibrium fluctuation trajectories provide the raw data necessary to determine time-correlation functions and probability distributions of coordinate displacements, which characterize conformational transitions of the DsRed complex.

Conformation and electronic population transfer in membrane-supported self-assembled porphyrin dimers by 2D fluorescence spectroscopy.

Two-dimensional fluorescence spectroscopy (2D FS) is applied to determine the conformation and femtosecond electronic population transfer in a dimer of magnesium meso tetraphenylporphyrin. The dimers are prepared by self-assembly of the monomer within the amphiphilic regions of 1,2-distearoyl-sn-glycero-3-phosphocholine liposomes. A theoretical framework to describe 2D FS experiments is presented, and a direct comparison is made between the observables of this measurement and those of 2D electronic spectroscopy (2D ES). The sensitivity of the method to varying dimer conformation is explored. A global multivariable fitting analysis of linear and 2D FS data indicates that the dimer adopts a "bent T-shaped" conformation. Moreover, the manifold of singly excited excitons undergoes rapid electronic dephasing and downhill population transfer on the time scale of ∼95 fs. The open conformation of the dimer suggests that its self-assembly is favored by an increase in entropy of the local membrane environment.

Fourier imaging correlation spectroscopy for cellular structure-function.

Experiments that optically probe the dynamics of intracellular species, including the center-of-mass displacements and internal conformational transitions of biological macromolecules, have the potential to study mechanisms of biochemical processes in living cells. This chapter reviews Fourier imaging correlation spectroscopy (FICS), a novel phase-selective approach to fluorescence fluctuation spectroscopy that measures the collective coordinate fluctuations from a large population of fluorescent species (N approximately 10(6)). In FICS experiments, a spatially modulated optical grating excites a fluorescently labeled sample. Phase-synchronous detection of the fluorescence, with respect to the phase of the exciting optical grating, can be used to monitor the fluctuations of partially averaged spatial coordinates. From these data are constructed two-point space-time correlation functions and probability distributions. FICS achieves a unique balance between signal-to-noise and signal information content. It represents a route to elevate signal levels, while acquiring detailed information about molecular coordinate trajectories.

Fluorescence-detected two-dimensional electronic coherence spectroscopy by acousto-optic phase modulation.

Two-dimensional electronic coherence spectroscopy (ECS) is an important method to study the coupling between distinct optical modes of a material system. Such studies often involve excitation using a sequence of phased ultrashort laser pulses. In conventional approaches, the delays between pulse temporal envelopes must be precisely monitored or maintained. Here, we introduce a new experimental scheme for phase-selective nonlinear ECS, which combines acousto-optic phase modulation with ultrashort laser excitation to produce intensity modulated nonlinear fluorescence signals. We isolate specific nonlinear signal contributions by synchronous detection, with respect to appropriately constructed references. Our method effectively decouples the relative temporal phases from the pulse envelopes of a collinear train of four sequential pulses. We thus achieve a robust and high signal-to-noise scheme for phase-selective ECS to investigate the resonant nonlinear optical response of photoluminescent systems. We demonstrate the validity of our method using a model quantum three-level system-atomic Rb vapor. Moreover, we show how our measurements determine the resonant complex-valued third-order susceptibility.