Total Internal Reflection Fluorescence Correlation Spectroscopy Research Articles

Quantification of membrane fluidity in bacteria using TIR-FCS

Plasma membrane fluidity is an important phenotypic feature that regulates the diffusion, function, and folding of transmembrane and membrane-associated proteins. In bacterial cells, variations in membrane fluidity are known to affect respiration, transport, and antibiotic resistance. Membrane fluidity must therefore be tightly regulated to adapt to environmental variations and stresses such as temperature fluctuations or osmotic shocks. Quantitative investigation of bacterial membrane fluidity has been, however, limited due to the lack of available tools, primarily due to the small size and membrane curvature of bacteria that preclude most conventional analysis methods used in eukaryotes. Here, we develop an assay based on total internal reflection-fluorescence correlation spectroscopy (TIR-FCS) to directly measure membrane fluidity in live bacteria via the diffusivity of fluorescent membrane markers. With simulations validated by experiments, we could determine how the small size, high curvature, and geometry of bacteria affect diffusion measurements and correct subsequent measurements for unbiased diffusion coefficient estimation. We used this assay to quantify the fluidity of the cytoplasmic membranes of the Gram-positive bacteria Bacillus subtilis (rod-shaped) and Staphylococcus aureus (coccus) at high (37°C) and low (20°C) temperatures in a steady state and in response to a cold shock, caused by a shift from high to low temperature. The steady-state fluidity was lower at 20°C than at 37°C, yet differed between B. subtilis and S. aureus at 37°C. Upon cold shock, the membrane fluidity decreased further below the steady-state fluidity at 20°C and recovered within 30 min in both bacterial species. Our minimally invasive assay opens up exciting perspectives for the study of a wide range of phenomena affecting the bacterial membrane, from disruption by chemicals or antibiotics to viral infection or change in nutrient availability.

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.

A total internal reflection-fluorescence correlation spectroscopy setup with pulsed diode laser excitation.

Fluorescence correlation spectroscopy (FCS) measures fluctuations in a (sub-)femtoliter volume to analyze the diffusive behavior of fluorescent particles. This highly sensitive method has proven to be useful for the analysis of dynamic biological systems as well as in chemistry, physics, and material sciences. It is routinely performed with commercial fluorescence microscopes, which provide a confined observation volume by the confocal technique. The evanescent wave of total internal reflectance (TIR) is used in home-built systems to permit a surface sensitive FCS analysis. We present a combined confocal and TIR-FCS setup which uses economic low-power pulsed diode lasers for excitation. Excitation and detection are coupled to time-correlated photon counting hardware. This allows simultaneous fluorescence lifetime and FCS measurements in a surface-sensitive mode. Moreover, the setup supports fluorescence lifetime correlation spectroscopy at surfaces. The excitation can be easily switched between TIR and epi-illumination to compare the surface properties with those in liquid bulk. The capabilities of the presented setup are demonstrated by measuring the diffusion coefficients of a free dye molecule, a labeled polyethylene glycol, and a fluorescent nanoparticle in confocal as well as in TIR-FCS.

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.

Plasma Membrane Organization of Epidermal Growth Factor Receptor in Resting and Ligand-Bound States

The spatial arrangement of the epidermal growth factor receptor (EGFR) on the cellular plasma membrane is one of the prime factors that control its downstream signaling pathways and related functions. However, the molecular organization, which spans the scale from nanometers to micrometer-size clusters, has not been resolved in detail, mainly due to a lack of techniques with the required spatiotemporal resolution. Therefore, we used imaging total internal reflection-fluorescence correlation spectroscopy to investigate EGFR dynamics on live CHO-K1 plasma membranes in resting and ligand-bound states. In combination with the fluorescence correlation spectroscopy diffusion law, this provides information on the subresolution organization of EGFR on cell membranes. We found that overall EGFR organization is sensitive to both cholesterol and the actin cytoskeleton. EGFR in the resting state is partly trapped in cholesterol-containing domains, whereas another fraction exhibits cholesterol independent trapping on the membrane. Disruption of the cytoskeleton leads to a broader range of EGFR diffusion coefficients and a reduction of hop diffusion. In the ligand-bound state we found a dose-dependent behavior. At 10 ng/mL EGF the EGFR is endocytosed and recycled to the membrane, whereas diffusion and organization do not change significantly. At 100 ng/mL EGF the EGFR forms clusters, which are subsequently internalized, whereas outside the clusters diffusivity increases and the organization of the receptor remains unchanged. After disruption of cholesterol-containing domains or actin cytoskeleton, EGF induces microscopic EGFR clusters on the membrane and endocytosis is inhibited.

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.

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.

Mapping CEACAM1 Dynamics and Interactions in Live Cells by Quantitative Fluorescence Techniques

Belonging to the immunoglobulin superfamily of membrane glycoproteins, carcinoembryonic antigen-related cell adhesion molecules (CEACAMs) are involved in diverse range of cellular processes and disease mechanisms through both homotypic and heterotypic interactions, In particular, CEACAM1 has been found to suppress tumorigenesis, hepatic lipogenesis, and systemic inflammation, but interestingly promotes tumour metastasis, angiogenesis, insulin clearance, and bacterial invasion. Molecular level insights into CEACAM1's behaviour are therefore essential for understanding its structure-function relationships. To this end, we have exploited high spatial- and temporal-resolution fluorescence imaging to elucidate the molecular dynamics and interactions of YFP-tagged CEACAM1 transfected into wild-type HeLa cells. Total internal reflection-fluorescence correlation spectroscopy (ITIR-FCS) enabled direct determination of the local diffusion coefficients of CEACAM1 in different regions of the cell membrane while number and brightness analysis (N&B) and spatial intensity distribution analysis (SpIDA) provided insights into the oligomeric state of CEACAM1. These strategies for characterizing the dynamics of transmembrane receptors in live cells show clear promise for probing glycoprotein function in normal and disease states.

Note: An easy way to enable total internal reflection-fluorescence correlation spectroscopy (TIR-FCS) by combining commercial devices for FCS and TIR microscopy

Total internal reflection-fluorescence correlation spectroscopy (TIR-FCS) is a powerful method for studying dynamic processes at liquid-solid interfaces that may have numerous applications in biology, physics, and material science. Despite of its power and versatility, however, the use of TIR-FCS is still rather limited. The main reason for this is the need of a complex, in-house constructed optical setup whose assembly and adjustment is a quite difficult task. Clearly, the availability of ready to use, commercial TIR-FCS setups will strongly boost the application of this important method in many research areas. In this note we show that although such setups are still not available in the market, a proper combination of commercial devices for confocal fluorescence correlation spectroscopy and for total internal reflection microscopy may enable TIR-FCS in a way that do not require any special optical alignments. Furthermore, we demonstrate the capabilities of the setup by measuring the diffusion coefficient of single dye molecule and quantum dots in the very proximity of a water-glass interface.

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.

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.

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.

Imaging Total Internal Reflection Fluorescence Cross-Correlation Spectroscopy (ITIR-FCCS)

Fluorescence correlation spectroscopy has been widely used to understand diffusion, flow processes and dynamics of reactions. Conventional FCS instruments use point detectors thus making multiplexing difficult. EMCCD based detection combined with TIRF illumination allows simultaneous detection of thousands of points. It has been demonstrated that this method, called Imaging Total Internal Reflection-Fluorescence Correlation Spectroscopy (ITIR- FCS), reduces background noise and allows the measurement of correlation functions on each pixel independently. This permits for the first time to observe molecular dynamics on a whole cell membrane simultaneously. This method has been applied to study diffusion phenomena in 2D lipid bilayers and cell membranes.In this work, ITIR-FCS is extended to perform spatial cross-correlations and is referred to as ITIR-FCCS. ITIR-FCCS is used to study flow and diffusion phenomena. Flow properties have been studied by a variety of techniques with suitable spatial resolution. In many of the techniques, there is a trade off between spatial (ICS) and temporal (FCS) resolution. ITIR- FCCS serves as a bridge between these two extremes with a spatial resolution of ∼ 300 nm and a temporal resolution of ∼ 0.4 ms.A generalized expression for ITIR-FCCS, allowing the cross-correlation of areas of any size and shape and for diffusion and flow, is derived. Since the expression contains only 5 fit parameters (flow velocities in x and y-direction, diffusion coefficient, concentration, size of the point spread function of the microscope) it is potentially calibration-free. This method was applied to three different systems: Diffusion, Flow, Diffusion and flow. Upon data analysis, flow velocities and diffusion coefficients are determined for all three systems and advantages and limitations are discussed. This technique is a promising tool for the analysis of diffusion and directed motion in cells with good spatial and temporal resolution.

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.

Multipoint fluorescence correlation spectroscopy with total internal reflection fluorescence microscope

We report simultaneous determination of diffusion coefficients at different points of a cell membrane using a multipoint fluorescence correlation spectroscopy (FCS) system. A system carrying seven detection areas in the evanescent field is achieved by using seven optical fibers on the image plane in the detection port of an objective-type total internal reflection FCS (TIR-FCS) system. Fluctuation of fluorescence intensity is monitored and evaluated using seven photomultiplier tubes (PMTs) and a newly constructed multichannel correlator. We demonstrate simultaneous-multipoint FCS, with a 3-mus time resolution, to investigate heterogeneous structures such as cell membranes and membrane-binding molecular dynamics near glass surfaces in live cells.

Vesicle Diffusion Close to a Membrane: Intermembrane Interactions Measured with Fluorescence Correlation Spectroscopy

The protein machinery controlling membrane fusion (or fission) has been well studied; however, the role of vesicle diffusion near membranes in these critical processes remains unclear. We experimentally and theoretically investigated the dynamics of small vesicles (∼50nm in diameter) that are diffusing near supported planar bilayers acting as “target” membranes. Using total internal reflection-fluorescence correlation spectroscopy, we examined the validity of theoretical analyses of vesicle–membrane interactions. Vesicles were hindered by hydrodynamic drag as a function of their proximity to the planar bilayer. The population distributions and diffusion kinetics of the vesicles were further affected by changing the ionic strength and pH of the buffer, as well as the lipid composition of the planar membrane. Effective surface charges on neutral bilayers were also analyzed by comparing experimental and theoretical data, and we show the possibility that vesicle dynamics can be modified by surface charge redistribution of the planar bilayer. Based on these results, we hypothesize that the dynamics of small vesicles, diffusing close to biomembranes, may be spatially restricted by altering local physiological conditions (e.g., salt concentration, lipid composition, and pH), which may represent an additional mechanism for controlling fusion (or fission) dynamics.

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.

Protein–surfactant interactions at hydrophobic interfaces studied with total internal reflection fluorescence correlation spectroscopy (TIR-FCS)

The aim of this work was to study the dynamics of proteins near solid surfaces in the presence or absence of competing surfactants by means of total internal reflection fluorescence correlation spectroscopy (TIR-FCS). Two different proteins were studied, bovine serum albumin (BSA) and Thermomyces lanuginosus lipase (TLL). A nonionic/anionic (C 12E 6/LAS) surfactant composition was used to mimic a detergent formulation and the surfaces used were C18 terminated glass. It was found that with increasing surfactant concentrations the term in the autocorrelation function (ACF) representing surface binding decreased. This suggested that the proteins were competed off the hydrophobic surface by the surfactant. When fitting the measured ACF to a model for surface kinetics, it was seen that with raised C 12E 6/LAS concentration, the surface interaction rate increased for both proteins. Under these experimental conditions this meant that the time the protein was bound to the surface decreased. At 10 μM C 12E 6/LAS the surface interaction was not visible for BSA, whereas it was still distinguishable in the ACF for TLL. This indicated that TLL had a higher affinity than BSA for the C18 surface. The study showed that TIR-FCS provides a useful tool to quantify the surfactant effect on proteins adsorption.

Total internal reflection with fluorescence correlation spectroscopy

Total internal reflection-fluorescence correlation spectroscopy (TIR-FCS) is an emerging technique that is used to measure events at or near an interface, including local fluorophore concentrations, local translational mobilities and the kinetic rate constants that describe the association and dissociation of fluorophores at the interface. TIR-FCS is also an extremely promising method for studying dynamics at or near the basal membranes of living cells. This protocol gives a general overview of the steps necessary to construct and test a TIR-FCS system using either through-prism or through-objective internal reflection geometry adapted for FCS. The expected forms of the autocorrelation function are discussed for the cases in which fluorescent molecules in solution diffuse through the depth of the evanescent field, but do not bind to the surface of interest, and in which reversible binding to the surface also occurs.