The pregnane X receptor (PXR), a member of the nuclear receptor superfamily, regulates the expression of drug-metabolizing enzymes in a ligand-dependent manner. The conventional view of nuclear receptor action is that ligand binding enhances the receptor’s affinity for coactivator proteins, while decreasing its affinity for corepressors. To date, however, no known rigorous biophysical studies have been conducted to investigate the interaction among PXR, its coregulators, and ligands. In this work, steady-state total internal reflection fluorescence microscopy (TIRFM) and total internal reflection with fluorescence recovery after photobleaching were used to measure the thermodynamics and kinetics of the interaction between the PXR ligand binding domain and a peptide fragment of the steroid receptor coactivator-1 (SRC-1) in the presence and absence of the established PXR agonist, rifampicin. Equilibrium dissociation and dissociation rate constants of ∼5 μM and ∼2 s–1, respectively, were obtained in the presence and absence of rifampicin, indicating that the ligand does not enhance the affinity of the PXR and SRC-1 fragments. Additionally, TIRFM was used to examine the interaction between PXR and a peptide fragment of the corepressor protein, the silencing mediator for retinoid and thyroid receptors (SMRT). An equilibrium dissociation constant of ∼70 μM was obtained for SMRT in the presence and absence of rifampicin. These results strongly suggest that the mechanism of ligand-dependent activation in PXR differs significantly from that seen in many other nuclear receptors.

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.

Fluorescence recovery after photobleaching and fluorescence correlation spectroscopy are the primary means for studying translational diffusion in biological systems. Both techniques, however, present numerous obstacles for measuring translational mobility in structures only slightly larger than optical resolution. We report a new method using through-prism total internal reflection fluorescence microscopy with continuous photobleaching to overcome these obstacles. Small structures, such as prokaryotic cells or isolated eukaryotic organelles, containing fluorescent molecules are adhered to a surface. This surface is continuously illuminated by an evanescent wave created by total internal reflection. The characteristic length describing the decay of the evanescent intensity with distance from the surface is smaller than the structures. The fluorescence decay rate resulting from continuous evanescent illumination is monitored as a function of the excitation intensity. The data at higher excitation intensities provide apparent translational diffusion coefficients for the fluorescent molecules within the structures because the decay results from two competing processes (the intrinsic photobleaching propensity and diffusion in the small structures). We present the theoretical basis for the technique and demonstrate its applicability by measuring the diffusion coefficient, 6.3 ± 1.1 μm2/s, of green fluorescent protein in Escherichia coli cells.

Although total internal reflection fluorescence microscopy (TIRFM)1, 2, 3, 4, 5, 6, 7 and fluorescence correlation spectroscopy (FCS)8, 9, 10, 11, 12 are mature techniques, the combination of the two is a relatively new method for studying surface-associated processes13. In TIRFM, an incident light beam propagates through a transparent solid and encounters a planar solid/liquid interface at an angle sufficient to induce TIR, which creates an electromagnetic field called the evanescent wave that propagates parallel to the solid/solution interface. The evanescent wave penetrates into the liquid medium and its intensity decays exponentially with distance from the surface, selectively exciting fluorescently labeled molecules along the interface. TIRFM is commonly used, singularly, to measure surface densities of bound, fluorescent molecules and to image cell–substrate contacts. TIRFM can also be used in conjunction with other techniques such as fluorescence recovery after photobleaching, fluorescence resonance energy transfer and fluorescence polarization to measure surface binding kinetic constants and translational diffusion coefficients, molecular distance-dependent events and population orientation distributions, respectively. In FCS, temporal fluctuations in fluorescence are correlated to obtain information about the processes giving rise to the observed fluctuations, when the number of molecules in the observation volume is small. The fluctuations typically arise from molecules diffusing into and out of the observation region or from molecules undergoing transitions between different fluorescent states. FCS is commonly used to measure equilibrium binding constants, diffusion coefficients, kinetic rate constants and oligomerization. In combination, TIR-FCS is a new, powerful tool for elucidating events that occur at or very close to surface/solution interfaces, such as the kinetic rate constants that describe the reversible association of fluorophores with the interface, local fluorophore concentrations and local translational mobilities. These parameters can be extracted from the magnitude and the rate and shape of the decay of the normalized fluorescence fluctuation autocorrelation function G(τ)14, 15, 16, 17, 18.In TIR-FCS, the most common method for generating TIR to date has been to use a prism, mounted to the stage of an inverted fluorescence microscope, through which a laser beam is loosely focused at the point of internal reflection (Fig. 1). The prism is optically coupled, generally by using a refractive index matching liquid such as glycerol, to the surface of interest. A surface, solution and coverslip sandwich is formed using spacers (Fig. 2). A high numerical aperture objective is coupled to the coverslip using immersion oil or water and is used to collect the evanescently excited fluorescence. The collected light is passed through a barrier filter (and usually a dichroic mirror) in the microscope to remove scattered radiation from the excitation source. A circular pinhole between 50 and 200 μm in diameter is placed in a confocal back-image plane to limit the area from which the fluorescence is collected (Fig. 1), and the remaining light is passed to a detector, which is typically a silicon avalanche photodiode or a photomultiplier. Alternatively, an optical fiber can be used in the same manner as a pinhole to limit the amount of light that reaches the detector19, 20, 21, 22.A second method for generating an evanescent field along a surface of interest is to use through-objective TIRFM. As the name implies, internal reflection is generated by focusing a laser beam through the periphery of a high numerical aperture objective, which is directly coupled to the surface of interest (Figs. 1 and 2), in a manner similar to epi-illumination. The evanescently excited fluorescence is collected back through the same objective, passes through a dichroic mirror and barrier filter to remove scattered excitation light and is passed through a pinhole to the detector. Additional focusing lenses may also be required to direct the light source through the objective at an angle great enough for TIRFM. Recent work details the building and testing of an in-house through-objective TIR-FCS system19, 20. Additionally, several manufacturers provide TIRFM attachments for inverted confocal microscopes (see MATERIALS), which can be adapted for FCS. Through-objective TIRFM may become the predominant configuration in the future, because of its commercial availability.Three advantages of through-prism TIRFM are the versatility of optical alignment, a lower background signal arising from the fact that the incident beam does not enter the interior of the microscope and (in many cases) lower cost. This geometry is also readily amenable to constructing evanescent interference patterns23 and to the use of very high refractive index substrates24. Advantages of through-objective TIRFM include sample-top accessibility, although through-prism configurations allowing such accessibility have also been designed3, decreased stray laser radiation in regions exterior to the microscope owing to the enclosed nature of the system, and the fact that slightly more fluorescence is collected through the higher refractive index side of the interface compared with the lower index side25. Also, through-objective TIRFM often gives a slightly better image quality owing to proper matching of the objective with the coverslip and sample plane. Theoretical treatments for the optical characteristics of the evanescent wave for both geometries have been presented26, 27, 28.A number of studies have demonstrated applications of TIR-FCS. In an early work, kinetic rate constants were determined for the nonspecific, reversible adsorption of rhodamine-labeled IgG and insulin to serum albumin-coated fused silica slides29. More recently, TIR-FCS was used to measure the binding kinetics of fluorescently labeled IgG specifically and reversibly associating with the mouse Fc receptor FcγRII in substrate-supported planar model membranes30. TIR-FCS has also been used to determine the equilibrium association constants and kinetic association rates for the reversible adsorption of rhodamine to bare fused silica surfaces and fused silica surfaces derivatized with C-18 alkyl chains, which serve as mimics for supports commonly used in reversed-phase chromatography31, 32. In addition, TIR-FCS has been used to study molecular transport in sol–gel films33, to study the diffusion of fluorescently labeled IgG in close proximity to planar model membranes14, 34 and in studies that verified the mathematical approximations for the depth of the evanescent wave in TIRFM systems by examining the diffusion of fluorescein close to substrates19, 21, 35. Very recently, dual-color cross-correlation TIR-FCS has been demonstrated as a method for detecting weak molecular pairs in binding assays22, 36.To date, few studies have used TIR-FCS in live-cell investigations. As the technique becomes more widely adopted through commercial distribution, the number of studies that implement TIR-FCS to study processes that occur at or near basal plasma membranes on surface-adherent cells is expected to increase, as TIR-FCS circumnavigates one of the major limitations of studying processes in the membranes of living cells with solution-based FCS. In solution-based FCS, the detection volume extends through the membrane into the extracellular and intracellular regions and excites molecules, which diffuse near the membrane but may not be relevant to the desired process8. By using TIRFM, the excitation region is limited by the depth of the evanescent field such that only components in, on or very near the membrane are optically excited. The feasibility of TIR-FCS as a method for examining dynamic processes in live cells has very recently been demonstrated by measurement of the diffusion of several fluorescent components (EGFP, the fluorescent lipid probe R18 and farnesylated EGFP) near or bound to the basal membranes of HeLa or COS7 cells by using TIR-FCS21.Future directions for TIR-FCS include the use of more sophisticated methods for analyzing fluorescence fluctuation data (e.g., photon-counting histograms or high-order correlation) and the use of two-photon excitation. A number of new possibilities can be envisioned for systems combining TIR-FCS with fast imaging detectors36, 37, 38. For example, kinetic maps describing the reversible association of fluorescent, cytoplasmic molecules with sites on the interiors of the basal plasma membranes of adherent cells might be generated, in a manner similar to that previously demonstrated by combining TIR excitation and fluorescence photobleaching recovery39. It is also expected that microfluidic devices40, 41, 42 will increasingly be used for various purposes36, 37, including significantly decreasing overall data acquisition time by using different channels for different sample types and either multiple single-photon counting detectors or a fast imaging detector.An elegant, recent paper described the imaging of rhodamine-labeled cross-bridges in muscle fibers using metal film-enhanced TIR, which reduces the background fluorescence and the depth of the evanescent wave, improving imaging43. The use of metal film-enhanced TIRFM or of very high refractive index substrates along with FCS would enhance the ability to conduct depth-dependent studies in living cells and in model biophysical processes such as diffusion near a wall14, 34. To date, most receptor–ligand binding studies that have used TIR-FCS have been conducted on systems in which the receptor was immobilized at the surface/solution interface30. A generalization in which receptors are laterally mobile and both kinetic and lateral diffusion information is extracted by systematically altering the observation area would significantly increase the range of applicability of TIR-FCS. TIR-FCS can in theory provide kinetic information about nonfluorescent molecules in the presence of fluorescently labeled molecules that compete for the same binding sites15, but experimental verification of this theoretical prediction has not yet been demonstrated.As the field of single-molecule fluorescence spectroscopy continues to expand, it is worth noting that TIR-FCS is not a true single-molecule method, in the sense that the same molecule is observed over an extended period of time. Although studies in which single molecules are observed to bind to and dissociate from surfaces give more direct measures of surface residency times, TIR-FCS is less tedious and capable of giving a more readily accessible measure of average kinetic behavior. A thorough description of the use TIR and FCS (separately) in single-molecule fluorescence studies can be found in a recent book44.In this protocol, we describe how to construct a TIR-FCS system using either a through-objective or through-prism geometry. When following this procedure, the required equipment will vary depending on whether the apparatus is constructed completely in-house, by modifying an in-house microscope or by using commercially available instruments for TIR excitation and/or FCS. In addition, the expense and the time required for assembling a TIR-FCS apparatus is highly dependent on whether existent or commercial TIR or FCS equipment is modified, or the apparatus is built from individual components. After instrument assembly and testing, samples can be prepared and numerous autocorrelation functions obtained in a single day.Troubleshooting advice can be found in Table 1.For situations in which fluorescent molecules in solution diffuse through the depth of the evanescent illumination but do not bind to the surface of interest, the autocorrelation function should have the following form: where A is the solution concentration of fluorescent molecules, h is the projected radius of a circular back-image plane aperture onto the sample plane, d is the evanescent wave depth, D is the diffusion coefficient of the fluorescent molecules in solution and N A is the average number of molecules in the observation volume. An example of this type of measurement is shown in Figure 3. The magnitude of G(τ) should be inversely proportional to the solution concentration of fluorescent molecules, to the evanescent wave depth and to the observation area size. The characteristic rate of decay, R e, should decrease for smaller solution diffusion coefficients (which can be achieved by increasing the molecular size) and for thicker evanescent wave depths (which can be achieved by decreasing the incidence angle).Equation (1) is appropriate for a situation in which the x–y area of the observed volume is larger than the evanescent wave depth and in which concentration fluctuations dissipate primarily through motions perpendicular to the surface of interest. Generalizations for situations in which lateral motion contributes to concentration fluctuation dissipation are also available19. Furthermore, equation (1) does not include possible contributions from photochemical events: for example, triplet state involvement. These events usually occur in time ranges on the order of microseconds and are dependent on excitation intensity. If the obtained autocorrelation functions, cropped at 10 μs, do not change with the excitation intensity, photochemical contributions are negligible. For situations in which these effects cannot be ignored, theoretical treatments describing the manner in which the effects change the functional form of the autocorrelation function are available19, 51.For situations in which fluorescent molecules in solution diffuse through the depth of the evanescent illumination and do reversibly bind to the surface of interest, the functional form of the autocorrelation function is in general much more complex15, 16. However, under certain conditions30 where N C is the average number of fluorescent molecules bound to the surface within the observation area and k d is the intrinsic dissociation rate of bound, fluorescent ligands from receptors. The first term in equation (2) describes diffusion through the evanescent wave depth without surface binding. The second term describes longer time fluorescence fluctuations arising from temporary surface binding. An example of this type of measurement is shown in Figure 3. The following two qualitative features should be observed in G(τ) in cases where reversible surface binding occurs: (a) The magnitude of G(τ) should be lower (not higher), when surface binding sites are available, as compared with matched background samples. This result follows from the fact that when surface binding sites are present, more molecules on average are present in the observed volume. (b) G(τ) for samples containing receptor-binding sites should have long-time components reporting surface binding events that are not present in G(τ) for samples not containing receptors.

In this paper, the conceptual basis and experimental design of total internal reflection with fluorescence correlation spectroscopy (TIR-FCS) is described. The few applications to date of TIR-FCS to supported membranes are discussed, in addition to a variety of applications not directly involving supported membranes. Methods related, but not technically equivalent, to TIR-FCS are also summarized. Future directions for TIR-FCS are outlined.