Total Internal Reflection Fluorescence Correlation Research Articles

HDL-membrane-interactions are highly influenced by the target membrane-lipid composition.

Lipid transfer from lipoprotein particles to cells is essential for lipid homeostasis. High-density lipoprotein (HDL) particles are part in an important mechanism in cholesterol homeostasis in the body and are mainly captured by cell membrane-associated scavenger receptor class B type 1 (SR-B1) from the bloodstream. Recently we were able to demonstrate an additional concept of a receptor-independent interaction between lipoprotein particles and lipid membranes. The role of the different lipid composition, properties, and organisation of the target lipid membrane itself has not yet been investigated. This work addresses the question of how the interaction of HDL with synthetic membranes is regulated by varying the chain length, the degree of saturation as well as the binding groups of the head-tail region of the target membrane lipids. Total internal reflection microscopy and fluorescence correlation spectroscopy allowed us to demonstrate that target lipid membrane composed of ether lipids, significantly influences the preferred interaction region in phase separated membranes. In addition, when HDL particles were incubated with single-phase membranes containing ether lipids, we observed that the change in the diffusion coefficient of a fluorescent tracer lipid was significantly higher than for membranes without ether lipids. The receptor-independent interaction of lipoprotein particles with membranes is a fairly new concept and these new findings will help to refine the knowledge of lipoprotein particle biology as well the treatment in dyslipidaemia diseases.

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.

A Funneled Conformational Landscape Governs Flavivirus Fusion Peptide Interaction with Lipid Membranes.

During host cell infection by flaviviruses such as dengue and Zika, acidic pH within the endosome triggers a conformational change in the envelope protein on the outer surface of the virion. This results in exposure of the ∼15 residue fusion peptide (FP) region, freeing it to induce fusion between the viral and endosomal membranes. A better understanding of the conformational dynamics of the FP in the presence of membranes, and the basis for its selectivity for anionic lipid species present within the endosome, would facilitate its therapeutic targeting with antiviral drugs and antibodies. In this work, multiscale modeling, simulations, and free energy calculations (including a total of ∼75 μs of atomic-resolution sampling), combined with imaging total internal reflection fluorescence correlation spectroscopy experiments, were employed to investigate the mechanisms of interaction of FP variants with lipid bilayers. Wild-type FPs (in the presence or absence of a fluorescein isothiocyanate tag) were shown to possess a funneled conformational landscape governing their exit from solvent and penetration into the lipid phase and to exhibit an electrostatically favored >2-fold affinity for membranes containing anionic species over purely zwitterionic ones. Conversely, the landscape was abolished in a nonfunctional point mutant, leading to a 2-fold drop in host membrane affinity. Collectively, our data reveal how the highly conserved flavivirus FP has evolved to funnel its conformational space toward a maximally fusogenic state anchored within the endosomal membrane. Therapeutically targeting the accessible ensemble of FP conformations may represent a new, rational strategy for blocking viral infection.

Polymer Diffusion in the Interphase Between Surface and Solution.

Total internal reflection fluorescence correlation spectroscopy (TIR-FCS) is applied to study the self-diffusion of poly(ethylene glycol) solutions in the presence of weakly attractive interfaces. Glass coverslips modified with aminopropyl- and propyl-terminated silanes are used to study the influence of solid surfaces on polymer diffusion. A model of three phases of polymer diffusion allows to describe the experimental fluorescence autocorrelation functions. Besides the two-dimensional diffusion of adsorbed polymer on the substrate and three-dimensional free diffusion in bulk solution, a third diffusion time scale is observed with intermediate diffusion times. This retarded three-dimensional diffusion in the solution is assigned to the long-range effects of solid surfaces on diffusional dynamics of polymers. The respective diffusion constants show Rouse scaling ( D ∼ N-1), indicating a screening of hydrodynamic interactions by the presence of the surface. Hence, the presented TIR-FCS method proves to be a valuable tool to investigate the effect of surfaces on polymer diffusion beyond the first adsorbed polymer layer on the 100 nm length scale.

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.

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 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.

Epidermal Growth Factor Receptor Localization in Cell Membranes Investigated by Imaging FCS

The epidermal growth factor receptor (EGFR) is a receptor tyrosine kinase that plays essential roles in cell growth and proliferation. Despite many years of research there are still open questions about its mechanism of function. We have addressed earlier the question of EGFR dimerization as well as phosphorylation in live cells. Here we address in particular its distribution and dynamics on the cell membrane. The simultaneous elucidation of membrane organization and dynamics requires measurements with good spatiotemporal resolution. We recently introduced Imaging Total Internal Reflection Fluorescence Correlation Spectroscopy (ITIR-FCS) with time resolution down to 0.3 ms and diffraction-limited resolution to study whole membrane areas with single molecule sensitivity. ITIR-FCS can test the FCS diffusion laws and determine whether diffusion is free, takes place within a meshwork or is hindered by traps. We demonstrate the feasibility of this approach with various structured artificial bilayers. Advancing to live cell measurements, we compare the diffusive behavior of DiI (C12 and C18), a membrane marker for the fluid phase, GPI-GFP, a putative microdomain marker, transferring receptor-mCherry, a receptor interacting with the cytoskeleton, and the epidermal growth factor receptor-mRFP (EGFR-mRFP). We monitor the dynamics of the different molecules under conditions of cholesterol depletion (mbCD), cytoskeleton depolymerization (Latrunculin A), and EGF stimulation. EGFR clearly shows changes upon stimulation, implying stronger domain localization, with little sensitivity to depolymerization of the cytoskeleton and moderate sensitivity to cholesterol depletion.

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.

Fluorescence correlation spectroscopy: a review of biochemical and microfluidic applications.

Over the years fluorescence correlation spectroscopy (FCS) has proven to be a useful technique that has been utilized in several fields of study. Although FCS initially suffered from poor signal-to-noise ratios, the incorporation of confocal microscopy has overcome this drawback and transformed FCS into a sensitive technique with high figures of merit. In addition, tandem methods have evolved to include dual-color cross-correlation, total internal reflection fluorescence correlation, and fluorescence lifetime correlation spectroscopy combined with time-correlated single-photon counting. In this review, we discuss several applications of FSC for biochemical, microfluidic, and cellular investigations.

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.

Measuring Surface Binding Thermodynamics and Kinetics by using Total Internal Reflection with Fluorescence Correlation Spectroscopy: Practical Considerations

The combination of total internal reflection illumination and fluorescence correlation spectroscopy (TIR-FCS) is an emerging method useful for, among a number of things, measuring the thermodynamic and kinetic parameters describing the reversible association of fluorescently labeled ligands in solution with immobilized, nonfluorescent surface binding sites. However, there are many parameters (both instrumental and intrinsic to the interaction of interest) that determine the nature of the acquired fluorescence fluctuation autocorrelation functions. In this work, we define criteria necessary for successful measurements, and then systematically explore the parameter space to define conditions that meet the criteria. The work is intended to serve as a guide for experimental design; in other words, to provide a methodology to identify experimental conditions that will yield reliable values of the thermodynamic and kinetic parameters for a given interaction. In vitro experiments to verify the theoretical predictions will be also provided.

Measuring Surface Binding Thermodynamics and Kinetics by Using Total Internal Reflection with Fluorescence Correlation Spectroscopy: Practical Considerations

The combination of total internal reflection illumination and fluorescence correlation spectroscopy (TIR-FCS) is an emerging method useful for, among a number of things, measuring the thermodynamic and kinetic parameters describing the reversible association of fluorescently labeled ligands in solution with immobilized, nonfluorescent surface binding sites. However, there are many parameters (both instrumental and intrinsic to the interaction of interest) that determine the nature of the acquired fluorescence fluctuation autocorrelation functions. In this work, we define criteria necessary for successful measurements and then systematically explore the parameter space to define conditions that meet the criteria. The work is intended to serve as a guide for experimental design, in other words, to provide a methodology to identify experimental conditions that will yield reliable values of the thermodynamic and kinetic parameters for a given interaction.

Electrostatic interactions of fluorescent molecules with dielectric interfaces studied by total internal reflection fluorescence correlation spectroscopy.

Electrostatic interactions between dielectric surfaces and different fluorophores used in ultrasensitive fluorescence microscopy are investigated using objective-based Total Internal Reflection Fluorescence Correlation Spectroscopy (TIR-FCS). The interfacial dynamics of cationic rhodamine 123 and rhodamine 6G, anionic/dianionic fluorescein, zwitterionic rhodamine 110 and neutral ATTO 488 are monitored at various ionic strengths at physiological pH. As analyzed by means of the amplitude and time-evolution of the autocorrelation function, the fluorescent molecules experience electrostatic attraction or repulsion at the glass surface depending on their charges. Influences of the electrostatic interactions are also monitored through the triplet-state population and triplet relaxation time, including the amount of detected fluorescence or the count-rate-per-molecule parameter. These TIR-FCS results provide an increased understanding of how fluorophores are influenced by the microenvironment of a glass surface, and show a promising approach for characterizing electrostatic interactions at interfaces.

Triplet-State Investigations of Fluorescent Dyes at Dielectric Interfaces Using Total Internal Reflection Fluorescence Correlation Spectroscopy

The triplet-state kinetics of several fluorescent dyes used in ultrasensitive fluorescence microscopy are investigated using total internal reflection fluorescence correlation spectroscopy (TIR-FCS). A theoretical outline of the correlation analysis and the physical aspects of evanescent excitation and fluorescence emission at dielectric interfaces are given. From this analysis, the rates of intersystem crossing and triplet decay are deduced for fluorescein, ATTO 488, rhodamine 110, rhodamine 123, and rhodamine 6G in aqueous buffer solutions. All investigated dyes show slightly higher triplet rates at the dielectric interface compared to bulk solution measurements. We attribute this enhancement to possible modifications of the dyes' photophysical properties near a dielectric interface. In the case of rhodamine 6G, the impact of changes in the dye concentration, ionic strength of the solvent, and potassium iodide concentration are also investigated. This leads to a better understanding of the influences of dye-dye, dye-solvent, and dye-surface interactions on the increased triplet intersystem crossing and triplet decay rates. The study shows that analysis of triplet-state kinetics by TIR-FCS not only results in a better understanding of how the photophysical properties of the dyes are affected by the presence of an interface, but also provides a means for probing the microenvironment near dielectric interfaces.

Applicability Of An EM-CCD For Spatially Resolved TIR-FCS

Diffusion constants (DC) of surface near fluorescent particles can be measured by total internal reflection fluorescence correlation microscopy (TIR-FCS). The usage of EM-CCDs instead of photo diodes offers a high degree of parallelization and the possibility of extracting additional information by spatial cross-correlation (TIR image correlation spectroscopy, TIR-ICS).Since temporal autocorrelation functions of particle fluorescence critically depend on CCD parameters such as pixel size and geometry, binning, sampling rate, and gain, we explored systematically the performance of an EM-CCD as detector in TIR-ICS. We found that variations in the sample geometry can be well described by a structure term (ST). Whereas in axial direction the ST is described by evanescent field depth, the lateral extension of the detection volume was found to be well approximated by a Gaussian fit to the convolution of the CCD pixel geometry with the measured point-spread-function for single pixel read-out. For higher binning we empirically could show a linear relationship between the Gaussian approximation for the lateral ST and the size of the quadratic ROI on the CCD used for detection (binning), with a correction factor (slope) that is independent of the CCD chip used.To test the performance of CB TIR-ICS we measured diffusion coefficients (DC) and particle numbers (PN) of fluorescent probes of different sizes (Fluorospheres and GFP) at varying viscosities, concentrations, and sampling rates. This allowed calculating the resolution of the method expressed as the minimal relative resolvable difference in PNs or DCs. Distinguishing between different probe concentrations was possible with differences in PN of 30%. In contrast differences as low as 6% in DC could be distinguished at DC-to-sampling-frequency-ratios smaller than 0.5∗10ˆ4nmˆ2.This renders TIR-ICS suitable and ideal for measuring spatially resolved dynamics of proteins in viscous media such as in live cells.