Imaging Total Internal Reflection Fluorescence Correlation Spectroscopy Research Articles

To Hop or not to Hop: Exceptions in the FCS Diffusion Law

Diffusion obstacles in membranes have not been directly visualized because of fast membrane dynamics and the occurrence of subresolution molecular complexes. To understand the obstacle characteristics, mobility-based methods are often used as an indirect way of assessing the membrane structure. Molecular movement in biological plasma membranes is often characterized by anomalous diffusion, but the exact underlying mechanisms are still elusive. Imaging total internal reflection fluorescence correlation spectroscopy (ITIR-FCS) is a well-established mobility-based method that provides spatially resolved diffusion coefficient maps and is combined with FCS diffusion law analysis to examine subresolution membrane organization. In recent years, although FCS diffusion law analysis has been instrumental in providing new insights into the membrane structure below the optical diffraction limit, there are certain exceptions and anomalies that require further clarification. To this end, we correlate the membrane structural features imaged by atomic force microscopy (AFM) with the dynamics measured using ITIR-FCS. We perform ITIR-FCS measurements on supported lipid bilayers (SLBs) of various lipid compositions to characterize the anomalous diffusion of lipid molecules in distinct obstacle configurations, along with the high-resolution imaging of the membrane structures with AFM. Furthermore, we validate our experimental results by performing simulations on image grids with experimentally determined obstacle configurations. This study demonstrates that FCS diffusion law analysis is a powerful tool to determine membrane heterogeneities implied from dynamics measurements. Our results corroborate the commonly accepted interpretations of imaging FCS diffusion law analysis, and we show that exceptions happen when domains reach the percolation threshold in a biphasic membrane and a network of domains behaves rather like a meshwork, resulting in hop diffusion.

Plasma membrane asymmetry of lipid organization: fluorescence lifetime microscopy and correlation spectroscopy analysis

A fundamental feature of the eukaryotic cell membrane is the asymmetric arrangement of lipids in its two leaflets. A cell invests significant energy to maintain this asymmetry and uses it to regulate important biological processes, such as apoptosis and vesiculation. The dynamic coupling of the inner or cytoplasmic and outer or exofacial leaflets is a challenging open question in membrane biology. Here, we combined fluorescence lifetime imaging microscopy (FLIM) with imaging total internal reflection fluorescence correlation spectroscopy (ITIR-FCS) to differentiate the dynamics and organization of the two leaflets of live mammalian cells. We characterized the biophysical properties of fluorescent analogs of phosphatidylcholine, sphingomyelin, and phosphatidylserine in the plasma membrane of two mammalian cell lines (CHO-K1 and RBL-2H3). Because of their specific transverse membrane distribution, these probes allowed leaflet-specific investigation of the plasma membrane. We compared the results of the two methods having different temporal and spatial resolution. Fluorescence lifetimes of fluorescent lipid analogs were in ranges characteristic for the liquid ordered phase in the outer leaflet and for the liquid disordered phase in the inner leaflet. The observation of a more fluid inner leaflet was supported by free diffusion in the inner leaflet, with high average diffusion coefficients. The liquid ordered phase in the outer leaflet was accompanied by slower diffusion and diffusion with intermittent transient trapping. Our results show that the combination of FLIM and ITIR-FCS with specific fluorescent lipid analogs is a powerful tool for investigating lateral and transbilayer characteristics of plasma membrane in live cell lines.

The disordered plant dehydrin Lti30 protects the membrane during water-related stress by cross-linking lipids

Dehydrins are intrinsically disordered proteins, generally expressed in plants as a response to embryogenesis and water-related stress. Their suggested functions are in membrane stabilization and cell protection. All dehydrins contain at least one copy of the highly conserved K-segment, proposed to be a membrane-binding motif. The dehydrin Lti30 (Arabidopsis thaliana) is up-regulated during cold and drought stress conditions and comprises six K-segments, each with two adjacent histidines. Lti30 interacts with the membrane electrostatically via pH-dependent protonation of the histidines. In this work, we seek a molecular understanding of the membrane interaction mechanism of Lti30 by determining the diffusion and molecular organization of Lti30 on model membrane systems by imaging total internal reflection- fluorescence correlation spectroscopy (ITIR-FCS) and molecular dynamics (MD) simulations. The dependence of the diffusion coefficient explored by ITIR-FCS together with MD simulations yields insights into Lti30 binding, domain partitioning, and aggregation. The effect of Lti30 on membrane lipid diffusion was studied on fluorescently labeled supported lipid bilayers of different lipid compositions at mechanistically important pH conditions. In parallel, we compared the mode of diffusion for short individual K-segment peptides. The results indicate that Lti30 binds the lipid bilayer via electrostatics, which restricts the mobility of lipids and bound protein molecules. At low pH, Lti30 binding induced lipid microdomain formation as well as protein aggregation, which could be correlated with one another. Moreover, at physiological pH, Lti30 forms nanoscale aggregates when proximal to the membrane suggesting that Lti30 may protect the cell by "cross-linking" the membrane lipids.

SPT and Imaging FCS Provide Complementary Information on the Dynamics of Plasma Membrane Molecules

The dynamics of biomolecules in the plasma membrane is of fundamental importance to understanding cellular processes. Cellular signaling often starts with extracellular ligand binding to a membrane receptor, which then transduces an intracellular signal. Ligand binding and receptor-complex activation often involve a complex rearrangement of proteins in the membrane, which results in changes in diffusion properties. Two widely used methods to characterize biomolecular diffusion are single-particle tracking (SPT) and imaging total internal reflection fluorescence correlation spectroscopy (ITIR-FCS). Here, we compare the results of recovered diffusion coefficients and mean-square displacements of the two methods by simulations of free, domain-confined, or meshwork diffusion. We introduce, to our knowledge, a new method for the determination of confinement radii from ITIR-FCS data. We further establish and demonstrate simultaneous SPT/ITIR-FCS for direct comparison within living cells. Finally, we compare the results obtained by SPT and ITIR-FCS for the receptor tyrosine kinase MET. Our results show that SPT and ITIR-FCS yield complementary information on diffusion properties of biomolecules in cell membranes.

Plasma Membrane Diffusion Modes of FcεRI Receptor for Immunoglobin E Measured with Imaging Fluorescence Correlation Spectroscopy

Spatio-temporal organization of the plasma membrane plays a significant role in cell signaling. A prevailing view is that the plasma membrane spatially segregates into ordered lipid (Lo-like) nanodomains co-existing with disordered lipid (Ld-like) regions. Our laboratory, using super-resolution fluorescence imaging, previously showed that immunoglobin E bound to its receptor, FceRI, (IgE-FceRI complex) undergoes time-dependent redistribution on the membrane surface after multivalent ligand stimulation, upon which the receptor is phosphorylated by membrane-anchored Lyn kinase. A large body of evidence shows that this interaction of IgE-FceRI and Lyn occurs in the ordered regions of the plasma membrane enriched in cholesterol. We are examining the nature and dynamics of plasma membrane organization as experienced by IgE-FceRI in resting state and after antigen stimulation. We employ imaging total internal reflection fluorescence correlation spectroscopy (ITIR-FCS), which maps lateral diffusion with pixel resolution, to investigate the diffusion distribution of IgE-FceRI complexes on the ventral surface of live RBL cells in native and experimentally modulated (e.g., cholesterol depleted) conditions. We also conduct spot variation FCS (svFCS), which analyzes space-dependence of diffusion coefficients from the same set of ITIR-FCS data, to reveal the existence of nanoscopic obstacles (such as nanodomains) that affect the diffusion of IgE-FceRI complexes. These observations are compared with the diffusion behavior of protein markers that are known to prefer ordered or disordered regions of plasma membrane. These results, in conjunction with super-resolution imaging, which determines the size and density of these complexes, will provide a new level of insight into plasma membrane organization and its dynamic remodeling upon ligand-receptor stimulation.

Spatiotemporal mapping of diffusion dynamics and organization in plasma membranes

Imaging fluorescence correlation spectroscopy (FCS) and the related FCS diffusion law have been applied in recent years to investigate the diffusion modes of lipids and proteins in membranes. These efforts have provided new insights into the membrane structure below the optical diffraction limit, new information on the existence of lipid domains, and on the influence of the cytoskeleton on membrane dynamics. However, there has been no systematic study to evaluate how domain size, domain density, and the probe partition coefficient affect the resulting imaging FCS diffusion law parameters. Here, we characterize the effects of these factors on the FCS diffusion law through simulations and experiments on lipid bilayers and live cells. By segmenting images into smaller 7 × 7 pixel areas, we can evaluate the FCS diffusion law on areas smaller than 2 µm and thus provide detailed maps of information on the membrane structure and heterogeneity at this length scale. We support and extend this analysis by deriving a mathematical expression to calculate the mean squared displacement (MSDACF) from the autocorrelation function of imaging FCS, and demonstrate that the MSDACF plots depend on the existence of nanoscopic domains. Based on the results, we derive limits for the detection of domains depending on their size, density, and relative viscosity in comparison to the surroundings. Finally, we apply these measurements to bilayers and live cells using imaging total internal reflection FCS and single plane illumination microscopy FCS.

The Dynamics and Localization of the Epidermal Growth Factor Receptor (EGFR) on Live Cell Plasma Membrane Studied by ITIR-FCS

The Epidermal Growth Factor Receptor (EGFR), a prototypical receptor tyrosine kinase, plays an important role in cellular processes such as proliferation, migration and apoptosis. EGFR is known to be strongly regulated by its localization and the state of activation on the plasma membrane. However, the spatial distribution of EGFR on the plasma membrane has not been resolved in detail.Here, we use Imaging Total Internal Reflection Fluorescence Correlation Spectroscopy (ITIR-FCS) to investigate EGFR behavior on live cells before and after stimulation. ITIR-FCS allows monitoring EGFR dynamics on cell membranes and in combination with the FCS diffusion laws provides information on the nanoscopic EGFR organization.Our results indicate that EGFR is partly trapped in cholesterol sensitive domains and also couples to the cytoskeleton. Stimulation by EGF activates EGFR in a dose dependent manner. At low-dose stimulation (10 ng/ml), the membrane diffusion and confinement of EGFR remain unchanged and EGFR internalization is cholesterol independent and EGFR recycles back to the membrane. At high-dose EGF treatment (100 ng/ml), EGFR endocytosis is cholesterol dependent and cholesterol-depletion impairs receptor internalization. In addition, cholesterol-depletion with concomitant EGF stimulation leads to stronger confinement of the receptor, independent of the EGF dose, as clusters accumulate on the membrane. Furthermore, the actin cytoskeleton promotes EGFR internalization after ligand stimulation. Disruption of the actin cytoskeleton inhibits EGFR endocytosis and induces clustering of EGFR on the cell membrane. This study demonstrates the capabilities of ITIR-FCS to follow receptor stimulation events on live cells. It demonstrates not only that EGFR activation is cholesterol and cytoskeleton dependent but also that the activation pathway is EGF dose dependent, thus providing new insights into receptor dynamics and organization on live cells.

Tracking Ceacam1 Interactions and Dynamics with homo-FRET and Image Correlation Techniques

The carcinoembryonic antigen (CEA) family plays an important role in numerous normal and pathogenic processes related to cellular growth and differentiation. Crucial roles of various CEA-family members are well established while the effect of carcinoembryonic antigen-related cellular adhesion molecule 1 (CEACAM1) on immune-cell function has only recently been recognized. CEACAM1 is known to exist in both monomeric and dimeric states that are heterogeneously distributed at the cell surface. However, which form participates in cell-cell interactions and its related dynamics remain unknown. We report here details of CEACAM1's spatial distribution, oligomeric state, diffusional characteristics and dynamics as determined by live cell homo-fluorescence energy resonance transfer (homo-FRET) microscopy and imaging total internal reflection fluorescence correlation spectroscopy (ITIR-FCS).

Bayesian Analysis of Imaging FCS Investigates the Interaction of Monomeric HIAPP with Live Cell Membrane

Human islet amyloid polypeptide (hIAPP), co-secreted with insulin from pancreatic β cells, is linked to the pathogenesis of type II diabetes mellitus. While the mechanism of hIAPP-membrane interaction at aggregating concentrations of the peptide is well established, the same is not true for monomeric hIAPP. We used imaging total internal reflection-Fluorescence Correlation Spectroscopy (ITIR-FCS) to monitor the effect of monomeric hIAPP on live cell membrane dynamics. The complex interaction of hIAPP with the plasma membrane operates via formation of domains. After hIAPP addition, overall membrane diffusion first increases and then decreases along with concomitant formation and enlargement of the domains which encompass the entire membrane in a time dependent manner. However, the current limitation of ITIR-FCS data analysis does not allow the complete characterization of the temporal evolution of the domains. We implemented Bayesian inference tests which were previously applied to confocal FCS. This allowed us to observe three phases in the evolution of hIAPP-membrane interaction - i) Pre-nucleation phase (1-10 min) where diffusion is single component at most of the pixels and the diffusion coefficient increases monotonically. ii) Propagation phase (15-40 min) where the two-component model fits better for the pixels within the domains. The components exhibit distinct but constant diffusion coefficients but their molecular fraction change with the slow diffusing fraction increasing in time. iii) Saturation phase (40-60 min) where pixels within the domain fit better with single-component model. Therefore, we conclude that hIAPP instantaneous increases membrane diffusion. This is followed by the formation of diffusion-restricted domains that eventually slows down overall membrane dynamics. The novel Bayesian inference analysis of imaging FCS successfully resolved hIAPP-induced domain formation and concomitant modulation of plasma membrane dynamics, which was not explained by the conventional fitting routine.

Bayesian Total Internal Reflection Fluorescence Correlation Spectroscopy Reveals hIAPP-Induced Plasma Membrane Domain Organization in Live Cells

Amyloid fibril deposition of human islet amyloid polypeptide (hIAPP) in pancreatic islet cells is implicated in the pathogenesis of type II diabetes. A growing number of studies suggest that small peptide aggregates are cytotoxic via their interaction with the plasma membrane, which leads to membrane permeabilization or disruption. A recent study using imaging total internal reflection-fluorescence correlation spectroscopy (ITIR-FCS) showed that monomeric hIAPP induced the formation of cellular plasma membrane microdomains containing dense lipids, in addition to the modulation of membrane fluidity. However, the spatial organization of microdomains and their temporal evolution were only partially characterized due to limitations in the conventional analysis and interpretation of imaging FCS datasets. Here, we apply a previously developed Bayesian analysis procedure to ITIR-FCS data to resolve hIAPP-induced microdomain spatial organization and temporal dynamics. Our analysis enables the visualization of the temporal evolution of multiple diffusing species in the spatially heterogeneous cell membrane, lending support to the carpet model for the association mode of hIAPP aggregates with the plasma membrane. The presented Bayesian analysis procedure provides an automated and general approach to unbiased model-based interpretation of imaging FCS data, with broad applicability to resolving the heterogeneous spatial-temporal organization of biological membrane systems.

Temperature dependence of diffusion in model and live cell membranes characterized by imaging fluorescence correlation spectroscopy

The organization of the plasma membrane is regulated by the dynamic equilibrium between the liquid ordered (Lo) and liquid disordered (Ld) phases. The abundance of the Lo phase is assumed to be a consequence of the interaction between cholesterol and the other lipids, which are otherwise in either the Ld or gel (So) phase. The characteristic lipid packing in these phases results in significant differences in their respective lateral dynamics. In this study, imaging total internal reflection fluorescence correlation spectroscopy (ITIR-FCS) is applied to monitor the diffusion within supported lipid bilayers (SLBs) as functions of temperature and composition. We show that the temperature dependence of membrane lateral diffusion, which is parameterized by the Arrhenius activation energy (EArr), can resolve the sub-resolution phase behavior of lipid mixtures. The FCS diffusion law, a novel membrane heterogeneity ruler implemented in ITIR-FCS, is applied to show that the domains in the So–Ld phase are static and large while they are small and dynamic in the Lo–Ld phase. Diffusion measurements and the subsequent FCS diffusion law analyses at different temperatures show that the modulation in membrane dynamics at high temperature (313K) is a cumulative effect of domain melting and rigidity relaxation. Finally, we extend these studies to the plasma membranes of commonly used neuroblastoma, HeLa and fibroblast cells. The temperature dependence of membrane dynamics for neuroblastoma cells is significantly different from that of HeLa or fibroblast cells as the different cell types exhibit a high level of compositional heterogeneity.

Calibration and Limits of Camera‐Based Fluorescence Correlation Spectroscopy: A Supported Lipid Bilayer Study

Camera-based fluorescence correlation spectroscopy (FCS) approaches allow the measurement of thousands of contiguous points yielding excellent statistics and details of sample structure. Imaging total internal reflection FCS (ITIR-FCS) provides these measurements on lipid membranes. Herein, we determine the influence of the point spread function (PSF) of the optical system, the laser power used, and the time resolution of the camera on the accuracy of diffusion coefficient and concentration measurements. We demonstrate that the PSF can be accurately determined by ITIR-FCS and that the laser power and time resolution can be varied over a wide range with limited influence on the measurement of the diffusion coefficient whereas the concentration measurements are sensitive to changes in the measurement parameters. One advantage of ITIR-FCS is that the measurement of the PSF has to be performed only once for a given optical setup, in contrast to confocal FCS in which calibrations have to be performed at least once per measurement day. Using optimized experimental conditions we provide diffusion coefficients for over ten different lipid membranes consisting of one, two and three constituents, measured in over 200,000 individual correlation functions. Using software binning and thus the inherent advantage of ITIR-FCS of providing multiple observation areas in a single measurement we test the FCS diffusion law and show how they can be complemented by the local information provided by the difference in cross-correlation functions (ΔCCF). With the determination of the PSF by ITIR-FCS and the optimization of measurement conditions ITIR-FCS becomes a calibration-free method. This allows us to provide measurements of absolute diffusion coefficients for bilayers with different compositions, which were stable over many different bilayer preparations over a time of at least one year, using a single PSF calibration.

Imaging Fluorescence Correlation Spectroscopy Characterizes Membrane Dynamics and Organization

Imaging Total Internal Reflection Fluorescence Correlation Spectroscopy (ITIR-FCS), a camera based FCS approach, characterizes membrane dynamics by measuring the diffusion coefficient at every point on a membrane patch and accesses membrane organization by using the so-called ΔCCF approach in which the cross-correlation of neighboring pixels is used to characterize membrane heterogeneity. Here we discuss the various technical aspects of ITIR-FCS and ΔCCF and the influence of time resolution, measurement length, and laser power on the reliability of the results. We show that ITIR-FCS can unambiguously determine the point spread function of a system and that this measurement is essential to obtain proper absolute values for diffusion coefficients and concentrations. We apply ITIR-FCS first to a range of supported lipid bilayers with increasing complexity from single lipid bilayers to binary and ternary mixtures consisting of different phospholipids, sphingomyelin, and cholesterol. ITIR-FCS yields very consistent diffusion coefficients over dozens of bilayer preparations. We complement this bottom-up approach, whereby the complexity of an artificial membrane is increased step-by-step to approach the complexity of the cellular plasma membrane, by a top-down approach in which we characterize live cell membranes in their natural state and altered in composition by a range of biochemical approaches. Since a single ITIR-FCS measurement can be binned per software after the recording, it is ideal for testing the FCS diffusion laws, which determine the dependence of the diffusion coefficient on the size of the observation area, and to demonstrate the existence of lipid domains in ternary lipid mixtures and cell membranes.

Imaging Fluorescence Cross-Correlation Spectroscopy as a Tool to Study Cell-Membrane Organization

The structure of biological membranes has been investigated for many years. However, progress is hindered by the fact that putative domains are highly dynamic and their size is smaller than the optical diffraction limit and thus direct observations are difficult. Therefore, there is a need to develop new biophysical tools which can infer the existence of domains within membranes and can follow their development over time. We have introduced in the past a method called Imaging Total Internal Reflection-Fluorescence Correlation Spectroscopy (ITIR-FCS) using EMCCD or sCMOS cameras. ITIR-FCS allows the measurement of a large number (up to ∼0.5 million) correlation curves at contiguous locations on cell membranes of live cells with millisecond time resolution. The spatial information within the data can be used to obtain information on the structure and organization of the membranes. This is achieved by calculating differences between the forward and backward cross-correlations between adjacent pixels A and B (CCFAB - CCFBA) or A, B, and C (CCFAB - CCFCB). The results can be depicted as histograms referred to as ΔCCF distributions. In this work we conduct measurements on supported lipid bilayers and cell membranes and perform simulations to demonstrate how ΔCCF distributions change characteristically with membrane complexity and structure. In particular, we demonstrate that domains with sizes below the diffraction limit have a characteristic broadening effect on the ΔCCF distributions. As an example we show that changes in membrane structure and organization of live neuroblastoma cells can be followed over the time course of an hour or more by way of ΔCCF distributions. To deal with large amount of data collected we developed an open source software, ImFCS, to calculate and fit the auto- and cross-correlation functions and depict the results in an imaging format.

Imaging Total Internal Reflection Fluorescence Correlation Spectroscopy (ITIR-FCS) Detects Multiple Lipid Domains on Live Cell Membranes

The cell membrane consists of a large number of different lipid molecules, of membrane spanning and associated proteins and is mechanically linked to the cytoskeleton and the extracellular matrix. It exhibits a complicated, highly dynamic structure which reflects its large number of constituent components and which is essential for signalling and trafficking in live cells. This structure is thought to have different characteristic length scales which can range from tens to hundreds of nanometers. Thus new methods are needed which can provide information on membrane structures at these different length scales with high temporal resolution. Imaging Total Internal Reflection Fluorescence Correlation Spectroscopy (ITIR-FCS) provides fluorescence correlation measurements on each point of a whole cell membranes with diffraction limited resolution and a better than millisecond time resolution. In particular the differences between spatial cross-correlation measurements, so-called ΔCCF, allows to infer changes in membrane organization and in combination with the diffusion coefficients provides information about membrane dynamics, organization and their changes during drug treatment. Here, in a combination of simulations, measurements on pure and mixed lipid supported bilayers, and live cell membranes stained with domain markers, we show that cell membrane composition is a crucial determinant of the type of domains existing at any point in time. By using markers for liquid ordered (sphingolipid binding domain, SBD) and disordered (DiIC18) lipid phases and a range of membrane composition- and cytoskeleton-influencing agents (methyl-β-cyclodextrin, latrunculin A, NB-DNJ, fumonisin B1, sphingomyelinase) we demonstrate that we have at least two different, ceramide and sphingolipid dependent, domain types, and that membrane fluidity and organization are not necessarily correlated and exist over different length scales.

ImFCS: A software for Imaging FCS data analysis and visualization

The multiplexing of fluorescence correlation spectroscopy (FCS), especially in imaging FCS using fast, sensitive array detectors, requires the handling of large amounts of data. One can easily collect in excess of 100,000 FCS curves a day, too many to be treated manually. Therefore, ImFCS, an open-source software which relies on standard image files was developed and provides a wide range of options for the calculation of spatial and temporal auto- and cross-correlations, as well as differences in Cross-Correlation Functions (ΔCCF). ImFCS permits fitting of standard models to correlation functions and provides optimized histograms of fitted parameters. Applications include the measurement of diffusion and flow with Imaging Total Internal Reflection FCS (ITIR-FCS) and Single Plane Illumination Microscopy FCS (SPIM-FCS) in biologically relevant samples. As a compromise between ITIR-FCS and SPIM-FCS, we extend the applications to Imaging Variable Angle-FCS (IVA-FCS) where sub-critical oblique illumination provides sample sectioning close to the cover slide.

Molecular Diffusion Measurement in Lipid Bilayers over Wide Concentration Ranges: A Comparative Study

Molecular diffusion in biological membranes is a determining factor in cell signaling and cell function. In the past few decades, three main fluorescence spectroscopy techniques have emerged that are capable of measuring molecular diffusion in artificial and biological membranes at very different concentration ranges and spatial resolutions. The widely used methods of fluorescence recovery after photobleaching (FRAP) and single-particle tracking (SPT) can determine absolute diffusion coefficients at high (>100 microm(-2)) and very low surface concentrations (single-molecule level), respectively. Fluorescence correlation spectroscopy (FCS), on the other hand, is well-suited for the intermediate concentration range of about 0.1-100 microm(-2). However, FCS in general requires calibration with a standard dye of known diffusion coefficient, and yields only relative measurements with respect to the calibration. A variant of FCS, z-scan FCS, is calibration-free for membrane measurements, but requires several experiments at different well-controlled focusing positions. A recently established FCS method, electron-multiplying charge-coupled-device-based total internal reflection FCS (TIR-FCS), referred to here as imaging TIR-FCS (ITIR-FCS), is also independent of calibration standards, but to our knowledge no direct comparison between these different methods has been made. Herein, we seek to establish a comparison between FRAP, SPT, FCS, and ITIR-FCS by measuring the lateral diffusion coefficients in two model systems, namely, supported lipid bilayers and giant unilamellar vesicles.