[show abstract][hide abstract] ABSTRACT: Fluorescence fluctuation methods have become invaluable research tools for characterizing the molecular-level physical and chemical properties of complex systems, such as molecular concentrations, dynamics, and the stoichiometry of molecular interactions. However, information recovery via curve fitting analysis of fluctuation data is complicated by limited resolution and challenges associated with identifying accurate fit models. We introduce a new approach to fluorescence fluctuation spectroscopy that couples multi-modal fluorescence measurements with multi-modal global curve fitting analysis. This approach yields dramatically enhanced resolution and fitting model discrimination capabilities in fluctuation measurements. The resolution enhancement allows the concentration of a secondary species to be accurately measured even when it constitutes only a few percent of the molecules within a sample mixture, an important new capability that will allow accurate measurements of molecular concentrations and interaction stoichiometry of minor sample species that can be functionally important but difficult to measure experimentally. We demonstrate this capability using τFCS, a new fluctuation method which uses simultaneous global analysis of fluorescence correlation spectroscopy and fluorescence lifetime data, and show that τFCS can accurately recover the concentrations, diffusion coefficients, lifetimes, and molecular brightness values for a two component mixture over a wide range of relative concentrations.
PLoS ONE 01/2014; 9(2):e90456. · 3.73 Impact Factor
[show abstract][hide abstract] ABSTRACT: Abstract Advances in electron and fluorescence microscopy imaging are now providing molecular-level resolution of macromolecular and supramolecular dynamic self-organization. Nowhere has this information been more impactful than in understanding the increasing diversity of biomolecular misfolding diseases. The amyloid-β protein associated with Alzheimer’s disease and its congeners have now offered insight into both neurodegenerative disease as well as functional scaffolds for constructing bio-inspired nanomaterials. Here we discuss the exploitation of these methods within these dynamic networks of paracrystalline supramolecular phases, and define minimal nucleation events, resolve the templated propagation events, and reveal the complex dynamic molecular networks of these polymorphic assemblies.
[show abstract][hide abstract] ABSTRACT: Fluorescence correlation spectroscopy (FCS) and related fluctuation
spectroscopy and microscopy methods have become important research tools
that enable detailed investigations of the chemical and physical
properties of molecules and molecular systems in a variety of complex
environments. When analyzed successfully fluctuation measurements often
provide unique information that is otherwise difficult to measure, such
as molecular concentrations and interaction stoichiometry. However,
information recovery via curve fitting of fluctuation data can present
challenges due to limited resolution and/or problems with fitting model
verification. We discuss a new approach to fluctuation data analysis
coupling multi-modal fluorescence measurements and global analysis, and
demonstrate how this approach can provide enhanced sensitivity and
resolution in fluctuation measurements. We illustrate the approach using
a combination of FCS and fluorescence lifetime measurements, here called
τFCS, and demonstrate the capability to recover the concentration of
two independent molecular species in a two component mixture even when
the species have identical diffusion coefficients and molecular
brightness values. This work was partially supported by NSF grants
MCB0817966 and DMR0907435.
[show abstract][hide abstract] ABSTRACT: Fluorescence correlation spectroscopy (FCS) and related fluctuation spectroscopy and microscopy methods have become important research tools that enable detailed investigations of the chemical and physical properties of molecules and molecular systems in a variety of complex environments. Information recovery via curve fitting of fluctuation data can present complicating challenges due to limited resolution and/or problems with fitting model verification. We discuss a new approach to data analysis called τFCS that couples multiple modes of signal acquisition, here specifically FCS and fluorescence lifetimes, with global analysis. We demonstrate enhanced resolution using τFCS, including the capability to recover the concentration of both molecular species in a two-component mixture even when the species have identical diffusion coefficients and molecular brightness values, provided their fluorescent lifetimes are distinct. We also demonstrate how τFCS provides useful tools for model discrimination in FCS curve fitting.
Methods in enzymology 01/2013; 518C:145-173. · 1.90 Impact Factor
[show abstract][hide abstract] ABSTRACT: Recent evidence suggests that simple peptides can access diverse amphiphilic phases, and that these structures underlie the robust and widely distributed assemblies implicated in nearly 40 protein misfolding diseases. Here we exploit a minimal nucleating core of the Aβ peptide of Alzheimer's disease to map its morphologically accessible phases that include stable intermolecular molten particles, fibers, twisted and helical ribbons, and nanotubes. Analyses with both fluorescence lifetime imaging microscopy (FLIM) and transmission electron microscopy provide evidence for liquid-liquid phase separations, similar to the coexisting dilute and dense protein-rich liquid phases so critical for the liquid-solid transition in protein crystallization. We show that the observed particles are critical for transitions to the more ordered cross-β peptide phases, which are prevalent in all amyloid assemblies, and identify specific conditions that arrest assembly at the phase boundaries. We have identified a size dependence of the particles in order to transition to the para-crystalline phase and a width of the cross-β assemblies that defines the transition between twisted fibers and helically coiled ribbons. These experimental results reveal an interconnected network of increasing molecularly ordered cross-β transitions, greatly extending the initial computational models for cross-β assemblies.
[show abstract][hide abstract] ABSTRACT: We use Fluorescence Lifetime Imaging Microscopy (FLIM) and Second
Harmonic Imaging Microscopy (SHIM) to investigate the fundamental
molecular mechanisms responsible for nucleation and growth of
amyloidogenic-derived nanomaterials. The nanomaterials are assembled
from of Amyloid-β(16-22), specifically Ac-KLVFFAE-NH2, the
nucleating core of the Alzheimer's Amyloid-β protein. We describe
how FLIM and SHIM can be used to follow different nucleation pathways
and to quantify structural heterogeneities within these complex
nanomaterials. New evidence suggests that different structures emerge
from distinct nucleation pathways and these insights inform our
understanding of the peptide self-assembly mechanisms. We discuss these
insights in the context of a top down understanding of amyloidogenic
diseases, the bottom up control of functional nanomaterials and the
discovery of realtime structural indicators for nanofabrication
[show abstract][hide abstract] ABSTRACT: Access to native protein structure depends on precise polypeptide folding and assembly pathways. Identifying folding missteps that may lead to the nearly 40 protein misfolding diseases could feature prominently in the development of intervention strategies. Accordingly, we have investigated the earliest steps of assembly by the folding nucleus of the Alzheimer's disease Abeta peptide with real-time imaging and fluorescence correlation spectroscopy. These analyses reveal the immediate formation of large micrometer size clusters maintaining properties of intermolecular molten globules. These dynamic unstructured aggregates serve as the nucleating sites for amyloid growth and, as with native protein folding, appear important for backbone desolvation. The resulting amyloid nucleus however is able to template monomer addition from solution at rates from 2K peptides/s at millimolar peptide concentrations. This direct observation of amyloid assembly unifies several divergent models that currently exist for protein misfolding.
Journal of the American Chemical Society 05/2010; 132(18):6306-8. · 10.68 Impact Factor
[show abstract][hide abstract] ABSTRACT: Models used in fluorescence correlation spectroscopy (FCS) generally assume diffusion of a standard Fickian nature such that the mean square displacement (MSD,) of the molecular diffusion is linearly proportional to time, i.e. t. In complex systems the diffusion can be anomalous, which is commonly described via a power law dependence of the MSD, i.e. t^alpha. When measuring anomalous dynamics using FCS, the correlation functions are typically measured over a single lengthscale and the anomalous exponent, alpha, is recovered through curve fitting. The anomalous exponent accurately describes the time dependence of the diffusion over the measurement lengthscale, yet for the majority of experimental systems it has not yet been tested whether the dynamics predicted by the fit are actually observed over different lengthscales -- i.e. whether or not the assumed power law dynamics truly describe the system dynamics. We investigate using scanning FCS methods that simultaneously measure correlation functions over a range of lengthscales in order to determine how accurately the physical models describe the dynamics. We use simulations to test these methods and discuss their application for measuring drug delivery rates in biomedical hydrogels.
[show abstract][hide abstract] ABSTRACT: Nucleation and growth mechanisms in amyloid materials are resolved using single molecule fluorescence imaging and spectroscopy. Results identify an intermolecular molten globule state as a key intermediate of the nucleation pathway.
[show abstract][hide abstract] ABSTRACT: We have investigated classical nuclear localization sequence (NLS) mediated protein trafficking by measuring biomolecular dynamics within living cells using two-photon fluorescence correlation spectroscopy. By directly observing the behavior of specific molecules in their native cellular environment, it is possible to uncover functional details that are not apparent from traditional biochemical investigations or functional assays. We show that the intracellular mobility of NLS cargoes and their import receptor proteins, karyopherin-alpha and karyopherin-beta, can be robustly measured and that quantitative comparison of intracellular diffusion coefficients provides new insights into nuclear transport mechanisms. Import cargo complexes are assembled throughout the cytoplasm, and their diffusion is slower than predicted by molecular weight due to specific interactions. Analysis of NLS cargo diffusion in the cytoplasm indicates that these interactions are likely disrupted by NLS cargo binding. Our results suggest that delivery of import receptors and NLS cargoes to nuclear pores may complement selective translocation through the pores as a functional mechanism for regulating transport of proteins into the nucleus.
[show abstract][hide abstract] ABSTRACT: A pigment array has been constructed within a paracrystalline amyloid nanotube and Förster energy transfer along the nanotube surface has been demonstrated to self-assembled acceptor dyes.
Chemical Communications 01/2009; · 6.38 Impact Factor
[show abstract][hide abstract] ABSTRACT: Complex diffusive dynamics are often observed when one is investigating the mobility of macromolecules in living cells and other complex environments, yet the underlying physical or chemical causes of anomalous diffusion are often not fully understood and are thus a topic of ongoing research interest. Theoretical models capturing anomalous dynamics are widely used to analyze mobility data from fluorescence correlation spectroscopy and other experimental measurements, yet there is significant confusion regarding these models because published versions are not entirely consistent and in some cases do not appear to satisfy the diffusion equation. Further confusion is introduced through variations in how fitting parameters are reported. A clear definition of fitting parameters and their physical significance is essential for accurate interpretation of experimental data and comparison of results from different studies acquired under varied experimental conditions. This article aims to clarify the physical meaning of the time-dependent diffusion coefficients associated with commonly used fitting models to facilitate their use for investigating the underlying causes of anomalous diffusion. We discuss a propagator for anomalous diffusion that captures the power law dependence of the mean-square displacement and can be shown to rigorously satisfy the extended diffusion equation provided one correctly defines the time-dependent diffusion coefficient. We also clarify explicitly the relation between the time-dependent diffusion coefficient and fitting parameters in fluorescence correlation spectroscopy.
[show abstract][hide abstract] ABSTRACT: The application of fluorescent proteins in live cells has greatly improved our ability to study molecular mobility, which both reflects molecular function in live cells and reveals the properties of the local environment. Although measuring molecular mobility with fluorescent fusion proteins is powerful and convenient, certain experiments still require exogenous macromolecules to be loaded into cells. Cell viability provides a rough gauge of cellular damage following membrane permeabilization, but it is unknown how permeabilization will affect intracellular mobility. We have used fluorescence correlation spectroscopy to measure the intracellular dynamics of the enhanced green fluorescent protein (EGFP) in living human embryonic kidney (HEK) cells under conditions where the EGFP is either expressed or loaded using streptolysin O (SLO) permeabilization to determine how permeabilization effects mobility. We found that purified EGFP loaded with SLO has the same mobility as the expressed EGFP, while the mobility of the expressed EGFP after SLO permeabilization treatment becomes slightly slower. Our results indicate that SLO permeabilization is often accompanied by the loss of cellular soluble proteins to the surrounding medium, which explains the apparent decrease in diffusion rates following treatment. These measurements are also relevant to the role of molecular crowding in the intracellular mobility of proteins.
Journal of Biomedical Optics 01/2008; 13(3):031214. · 2.88 Impact Factor
[show abstract][hide abstract] ABSTRACT: Fluorescence correlation spectroscopy (FCS) has become an increasingly important measurement tool for biological and biomedical investigations, with the capability to assay molecular dynamics and interactions both in vitro and within living cells. Information recovery in FCS requires an accurate characterization and calibration of the observation volume. A number of recent reports have demonstrated that the calibration of the observation volume is excitation power dependent, a complication that arises due to excitation saturation. While quantitative models are now available to account for these volume variations, many researchers attempt to avoid saturation issues by working with low nonsaturating excitation intensities. For two-photon excited fluorescence, this is typically thought to be achievable by working with excitation powers for which the total measured fluorescence signal maintains its quadratic dependence on excitation intensity. We demonstrate that observing only the power dependence of the fluorescence intensity will tend to underestimate the importance of saturation, and explain these findings in terms of basic physical models.
Microscopy Research and Technique 09/2007; 70(8):682-6. · 1.59 Impact Factor
[show abstract][hide abstract] ABSTRACT: Messenger RNA transcripts are coated from cap to tail with a dynamic combination of RNA binding proteins that process, package, and ultimately regulate the fate of mature transcripts. One class of RNA binding proteins essential for multiple aspects of mRNA metabolism consists of the poly(A) binding proteins. Previous studies have concentrated on the canonical RNA recognition motif-containing poly(A) binding proteins as the sole family of poly(A)-specific RNA binding proteins. In this study, we present evidence for a previously uncharacterized poly(A) recognition motif consisting of tandem CCCH zinc fingers. We have probed the nucleic acid binding properties of a yeast protein, Nab2, that contains this zinc finger motif. Results of this study reveal that the seven tandem CCCH zinc fingers of Nab2 specifically bind to polyadenosine RNA with high affinity. Furthermore, we demonstrate that a human protein, ZC3H14, which contains CCCH zinc fingers homologous to those found in Nab2, also specifically binds polyadenosine RNA. Thus, we propose that these proteins are members of an evolutionarily conserved family of poly(A) RNA binding proteins that recognize poly(A) RNA through a fundamentally different mechanism than previously characterized RNA recognition motif-containing poly(A) binding proteins.
Proceedings of the National Academy of Sciences 08/2007; 104(30):12306-11. · 9.74 Impact Factor
[show abstract][hide abstract] ABSTRACT: While the growth and structure of amyloid fibers with ß-sheet secondary structure has been widely investigated in recent years, the mechanism of self-assembly remains poorly understood. Multiple intermediate species have been proposed to play important roles in the self assembly process, yet many of these remain poorly defined or have not been clearly observed. Fluorescence microscopy and spectroscopy should provide powerful tools to amyloid formation mechanisms, although given the tight packing of molecules within amyloid structures one must be concerned about the extent to which the coupling of fluorescent probes will interfere with the amyloid formation process. We have performed systematic characterization of the self assembly and interactions between a model amyloid forming peptide, residues 16-22 from the amyloid beta peptide, together with two different rhodamine conjugated forms of this same peptide sequence. We observe that in some cases, the fluorescent dye does appear to alter the morphology of assembled amyloid structures. We also report on amyloid formation using mixtures of labeled and unlabeled peptides which does not perturb the morphology of the amyloid fibers and tubes, and appears to provide an excellent system for further investigation of amyloid formation.
[show abstract][hide abstract] ABSTRACT: This chapter introduces to electronic cameras, discusses the various parameters considered for evaluating their performance, and describes some of the key features of different camera formats. The chapter also presents the basic understanding of functioning of the electronic cameras and how these properties can be exploited to optimize image quality under low-light conditions. Although there are many types of cameras available for microscopy, the most reliable type is the charge-coupled device (CCD) camera, which remains preferred for high-performance systems. If time resolution and frame rate are of no concern, slow-scan CCDs certainly offer the best available performance, both in terms of the signal-to-noise ratio and their spatial resolution. Slow-scan cameras are thus the first choice for experiments using fixed specimens such as measurements using immune fluorescence and fluorescence in situ hybridization. However, if video rate imaging is required, one need not evaluate slow-scan CCD cameras. A very basic video CCD may suffice if samples are heavily labeled or are not perturbed by high intensity illumination. When video rate imaging is required for very dim specimens, the electron multiplying CCD camera is probably the most appropriate at this technological stage. Intensified CCDs provide a unique tool for applications in which high-speed gating is required. The variable integration time video cameras are very attractive options if one needs to acquire images at video rate acquisition, as well as with longer integration times for less bright samples. This flexibility can facilitate many diverse applications with highly varied light levels.
Methods in cell biology 02/2007; 81:219-49. · 1.44 Impact Factor