Fourier Imaging Correlation Spectroscopy Research Articles

Subcellular Dynamics and Protein Conformation Fluctuations Measured by Fourier Imaging Correlation Spectroscopy

Novel high signal-to-noise spectroscopic experiments that probe the dynamics of microscopic objects have the potential to reveal complex intracellular biochemical mechanisms, or the slow relaxations of soft matter systems. This article reviews the implementation of Fourier imaging correlation spectroscopy (FICS), a phase-selective approach to fluorescence fluctuation spectroscopy that employs a unique route to elevate signal levels while acquiring detailed information about molecular coordinate trajectories. The review demonstrates the broad applicability of FICS by discussing two recent studies. The dynamics of Saccharomyces cerevisiae yeast mitochondria are characterized with FICS and provide detailed information about the influence of specific cytoskeletal elements on the movement of this organelle. In another set of experiments, polarization-modulated FICS captures conformational dynamics and molecular translational dynamics of the fluorescent protein DsRed, and analyses by four-point correlation and joint distribution functions of the corresponding data reveal statistically meaningful pathways of DsRed switching between different optical conformations.

Actin polymerization driven mitochondrial transport in mating S. cerevisiae

The dynamic microenvironment of cells depends on macromolecular architecture, equilibrium fluctuations, and nonequilibrium forces generated by cytoskeletal proteins. We studied the influence of these factors on the motions of mitochondria in mating S. cerevisiae using Fourier imaging correlation spectroscopy (FICS). Our measurements provide detailed length-scale dependent information about the dynamic behavior of mitochondria. We investigate the influence of the actin cytoskeleton on mitochondrial motion and make comparisons between conditions in which actin network assembly and disassembly is varied either by using disruptive pharmacological agents or mutations that alter the rates of actin polymerization. Under physiological conditions, nonequilibrium dynamics of the actin cytoskeleton leads to 1.5-fold enhancement of the long-time mitochondrial diffusion coefficient and a transient subdiffusive temporal scaling of the mean-square displacement (MSD proportional, variant tau (alpha), with alpha = 2/3). We find that nonequilibrium forces associated with actin polymerization are a predominant factor in driving mitochondrial transport. Moreover, our results lend support to an existing model in which these forces are directly coupled to mitochondrial membrane surfaces.

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.

Dynamics of Conformational Transitions in DsRed as Detected by Polarization-Modulated MFICS

Non-invasive spectroscopic approaches that are sensitive to fluctuations on the single molecule scale are desirable as methods to characterize the behaviour of proteins in living cells. Here we present molecular Fourier imaging correlation spectroscopy (MFICS) as a novel, high signal-to-noise approach to detect the time-varying translational movement and conformational transitions of the fluorescent protein DsRed. Thermally induced conformational transitions of the DsRed tetramer lead to changes in the optical dipolar coupling between adjacent chromophore sites, and our implementation of polarization- and intensity-modulated photo-excitation generates a phase sensitive signal that is separable into a component that comes from internal, thermally induced conformational transitions and a component which stems from translational diffusion. Based upon joint probability distributions and two-dimensional spectral densities of these measurements in conjunction with existing structural information, we interpret our measurement in terms of transitions between distinct energy-transfer-coupled conformations of DsRed.

Simultaneous measurement of fluorescence anisotropy and translational fluctuations by polarization-modulated MFICS

A novel, high signal-to-noise strategy is presented for phase-selective, wavevector- and polarization-resolved fluorescence fluctuation spectroscopy. The approach is based on molecular Fourier imaging correlation spectroscopy (MFICS) and combines polarization- and intensity-modulated photoexcitation with phase-synchronous detection to simultaneously monitor centre-of-mass and slow fluorescence anisotropy fluctuations from a relatively large number (∼106) of fluorescent molecules. The method is demonstrated using DsRed, a tetrameric complex of fluorescent protein subunits, to unambiguously separate signal contributions due to slow optical conformational fluctuations from translational diffusion.

Translational Diffusion of Fluorescent Proteins by Molecular Fourier Imaging Correlation Spectroscopy

The ability to noninvasively observe translational diffusion of proteins and protein complexes is important to many biophysical problems. We report high signal/noise (≥250) measurements of the translational diffusion in viscous solution of the fluorescent protein, DsRed. This is carried out using a new technique: molecular Fourier imaging correlation spectroscopy (M-FICS). M-FICS is an interferometric method that detects a collective Fourier component of the fluctuating density of a small population of fluorescent molecules, and provides information about the distribution of molecular diffusivities. A theoretical analysis is presented that expresses the detected signal fluctuations in terms of the relevant time-correlation functions for molecular translational diffusion. Furthermore, the role played by optical orientational degrees of freedom is established. We report Fickian self-diffusion of the DsRed tetramer at short timescales. The long-time deviation of our data from Fickian behavior is used to determine the variance of the distribution of the protein self-diffusion coefficient. We compare our results to the expected outcomes for 1), a bi-disperse distribution of protein species, and 2), dynamic disorder of the host solvent.

Direct measurement of relative and collective diffusion in a dilute binary colloidal suspension

Experimental characterization of the dynamics of multicomponent fluids is a problem of general importance to the field of complex fluids. We demonstrate a new experimental approach, termed two-color Fourier imaging correlation spectroscopy, which allows direct measurement of the partial dynamic structure factors, S(11)(k,tau), S(22)(k,tau), and S(12)(k,tau), where 1, 2 label the component species of a binary colloidal suspension. Linear combinations of the partial dynamic structure factors yield the characteristic time-correlation functions of the binary fluid. These are the correlation functions of concentration fluctuations S(CC)(k,tau), number density fluctuations S(NN)(k,tau), and cross-correlations between number density and concentration fluctuations S(NC)(k,tau). Test measurements are performed on a dilute symmetric mixture of fluorescently labeled 0.5 and 1.0 microm polystyrene spheres. From these data, we determine generalized collective and relative diffusion coefficients, and compare them to the predictions for an ideal mixture of noninteracting particles.

Cytoskeletal-assisted dynamics of the mitochondrial reticulum in living cells.

Subcellular organelle dynamics are strongly influenced by interactions with cytoskeletal filaments and their associated motor proteins, and lead to complex multiexponential relaxations that occur over a wide range of spatial and temporal scales. Here we report spatio-temporal measurements of the fluctuations of the mitochondrial reticulum in osteosarcoma cells by using Fourier imaging correlation spectroscopy, over time and distance scales of 10(-2) to 10(3) s and 0.5-2.5 microm. We show that the method allows a more complete description of mitochondrial dynamics, through the time- and length-scale-dependent collective diffusion coefficient D(k,tau), than available by other means. Addition of either nocodazole to disrupt microtubules or cytochalasin D to disassemble microfilaments simplifies the intermediate scattering function. When both drugs are used, the reticulum morphology of mitochondria is retained even though the cytoskeletal elements have been de-polymerized. The dynamics of the organelle are then primarily diffusive and can be modeled as a collection of friction points interconnected by elastic springs. This study quantitatively characterizes organelle dynamics in terms of collective cytoskeletal interactions in living cells.

Structure and dynamics of fluorescently labeled complex fluids by fourier imaging correlation spectroscopy

We present a method of Fourier imaging correlation spectroscopy (FICS) that performs phase-sensitive measurements of modulated optical signals from fluorescently labeled complex fluids. FICS experiments probe the time-dependent trajectory of a spatial Fourier component of the fluid particle density at a specified wave number k, and provide a direct route to the intermediate scattering function. The FICS approach overcomes signal sensitivity problems associated with dynamic light scattering, while offering a means to acquire time-dependent information about spatial distributions of fluorescent particles, superior in efficiency to direct imaging methods. We describe the instrumental setup necessary to perform FICS experiments, and outline the theory that establishes the connection between FICS observables and statistical mechanical quantities describing liquid state dynamics. Test measurements on monolayer suspensions of rhodamine labeled polystyrene spheres are detailed.

Dynamics of the Mitochondrial Reticulum in Live Cells using Fourier Imaging Correlation Spectroscopy and Digital Video Microscopy

We report detailed studies of the dynamics of the mitochondrial reticulum in live cells using two independent experimental techniques: Fourier imaging correlation spectroscopy and digital video fluorescence microscopy. When both methods are used to study the same system, it is possible to directly compare measurements of preaveraged statistical dynamical quantities with their microscopic counterparts. This approach allows the underlying mechanism of the observed rates to be determined. Our results indicate that the dynamics of the reticulum structure is composed of two independent contributions, each important on very different time and length scales. During short time intervals (1–15 sec), local regions of the reticulum primarily undergo constrained thermally activated motion. During long time intervals (>15 sec), local regions of the reticulum undergo long-range “jump” motions that are associated with the action of cytoskeletal filaments. Although the frequency of the jumps depend on the physiological state of the cells, the average jump distance (∼0.8 μm) is unaffected by metabolic activity. During short time intervals, the dynamics appear to be spatially heterogeneous, whereas the cumulative effect of the infrequent jumps leads to the appearance of diffusive motion in the limit of long time intervals.

Measurement of the dynamic structure function of fluorescently labeled complex fluids by fourier imaging correlation spectroscopy

Wave-number-resolved time-correlation functions of density fluctuations are determined from purely incoherent fluorescence signals using a new method, Fourier imaging correlation spectroscopy (FICS). We demonstrate the application of FICS to chemical and biological samples with ultrahigh signal sensitivity and time resolution superior to direct imaging methods. We outline the theory that establishes the connection between FICS observables and statistical mechanical quantities describing liquid state dynamics.