Measurement of single macromolecule orientation by total internal reflection fluorescence polarization microscopy

A new approach is presented for measuring the three-dimensional orientation of individual macromolecules using single molecule fluorescence polarization (SMFP) microscopy. The technique uses the unique polarizations of evanescent waves generated by total internal reflection to excite the dipole moment of individual fluorophores. To evaluate the new SMFP technique, single molecule orientation measurements from sparsely labeled F-actin are compared to ensemble-averaged orientation data from similarly prepared densely labeled F-actin. Standard deviations of the SMFP measurements taken at 40 ms time intervals indicate that the uncertainty for individual measurements of axial and azimuthal angles is approximately 10 degrees at 40 ms time resolution. Comparison with ensemble data shows there are no substantial systematic errors associated with the single molecule measurements. In addition to evaluating the technique, the data also provide a new measurement of the torsional rigidity of F-actin. These measurements support the smaller of two values of the torsional rigidity of F-actin previously reported.     

Visualizing single molecules inside living cells using total internal reflection fluorescence microscopy

Over the past 10 years, advances in laser and detector technologies have enabled single fluorophores to be visualized in aqueous solution. Here, we describe methods based on total internal reflection fluorescence microscopy (TIRFM) that we have developed to study the behavior of individual protein molecules within living mammalian cells. We have used cultured myoblasts that were transiently transfected with DNA plasmids encoding a target protein fused to green fluorescent protein (GFP). Expression levels were quantified from confocal images of control dilutions of GFP and cells with 1-100 nM GFP were then examined using TIRFM. An evanescent field was produced by a totally internally reflected, argon ion laser beam that illuminated a shallow region (50-100 nm deep) at the glass-water interface. Individual GFP-tagged proteins that entered the evanescent field appeared as individual, diffraction-limited spots of light, which were clearly resolved from background fluorescence. Molecules that bound to the basal cell membrane remained fixed in position for many seconds, whereas those diffusing freely in the cytoplasm disappeared within a few milliseconds. We developed automated detection and tracking methods to recognize and characterize the behavior of single molecules in recorded video sequences. This enabled us to measure the kinetics of photobleaching and lateral diffusion of membrane-bound molecules.     

Quantifying myosin light chain phosphorylation in single adherent cells with automated fluorescence microscopy

Background  

In anchorage dependent cells, myosin generated contractile forces affect events closely associated with adhesion such as the formation of stress fibers and focal adhesions, and temporally distal events such as entry of the cell into S-phase. As occurs in many signaling pathways, a phosphorylation reaction (in this case, phosphorylation of myosin light chain) is directly responsible for cell response. Western blotting has been useful in measuring intracellular phosphorylation events, but cells are lysed in the process of sample preparation for western blotting, and spatial information such as morphology, localization of the phosphorylated species, and the distribution of individual cell responses across the population is lost. We report here a reliable automated microscopy method for quantitative measurement of myosin light chain phosphorylation in adherent cells. This method allows us to concurrently examine cell morphology, cell-cell contact, and myosin light chain diphosphorylation in vascular smooth muscle cells.     

Three-dimensional characterization of tethered microspheres by total internal reflection fluorescence microscopy

Tethered particle microscopy is a powerful tool to study the dynamics of DNA molecules and DNA-protein complexes in single-molecule experiments. We demonstrate that stroboscopic total internal reflection microscopy can be used to characterize the three-dimensional spatiotemporal motion of DNA-tethered particles. By calculating characteristic measures such as symmetry and time constants of the motion, well-formed tethers can be distinguished from defective ones for which the motion is dominated by aberrant surface effects. This improves the reliability of measurements on tether dynamics. For instance, in observations of protein-mediated DNA looping, loop formation is distinguished from adsorption and other nonspecific events.     

Imaging constitutive exocytosis with total internal reflection fluorescence microscopy

Total internal reflection fluorescence microscopy has been applied to image the final stage of constitutive exocytosis, which is the fusion of single post-Golgi carriers with the plasma membrane. The use of a membrane protein tagged with green fluorescent protein allowed the kinetics of fusion to be followed with a time resolution of 30 frames/s. Quantitative analysis allowed carriers undergoing fusion to be easily distinguished from carriers moving perpendicularly to the plasma membrane. The flattening of the carriers into the plasma membrane is seen as a simultaneous rise in the total, peak, and width of the fluorescence intensity. The duration of this flattening process depends on the size of the carriers, distinguishing small spherical from large tubular carriers. The spread of the membrane protein into the plasma membrane upon fusion is diffusive. Mapping many fusion sites of a single cell reveals that there are no preferred sites for constitutive exocytosis in this system.     

Combined scanning probe and total internal reflection fluorescence microscopy

Combining scanning probe and optical microscopy represents a powerful approach for investigating structure-function relationships and dynamics of biomolecules and biomolecular assemblies, often in situ and in real-time. This platform technology allows us to obtain three-dimensional images of individual molecules with nanometer resolution, while simultaneously characterizing their structure and interactions though complementary techniques such as optical microscopy and spectroscopy. We describe herein the practical strategies for the coupling of scanning probe and total internal reflection fluorescence microscopy along with challenges and the potential applications of such platforms, with a particular focus on their application to the study of biomolecular interactions at membrane surfaces.     

Real-time measurements of actin filament polymerization by total internal reflection fluorescence microscopy

Understanding the mechanism of actin polymerization and its regulation by associated proteins requires an assay to monitor polymerization dynamics and filament topology simultaneously. The only assay meeting these criteria is total internal reflection fluorescence microscopy (Amann and Pollard, 2001; Fujiwara et al., 2002). The fluorescence signal is fourfold stronger with actin labeled on Cys-374 with Oregon green rather than rhodamine. To distinguish growth at barbed and pointed ends we used image drift correction and maximum intensity projections to reveal points where single N-ethylmaleimide inactivated myosins attach filaments to the glass coverslip. We estimated association rates at high actin concentrations and dissociation rates near and below the critical actin concentration. At the barbed end, the association rate constant for Mg-ATP-actin is 7.4 microM(-1) s(-1) and the dissociation rate constant is 0.89 s(-1). At the pointed end the association and dissociation rate constants are 0.56 microM(-1) s(-1) and 0.19 s(-1). When vitamin D binding protein sequesters all free monomers, ADP-actin dissociates from barbed ends at 1.4 s(-1) and from pointed ends at 0.16 s(-1) regardless of buffer nucleotide.     

Heterogeneous amylin fibril growth mechanisms imaged by total internal reflection fluorescence microscopy

Total internal reflection fluorescence microscopy has been used to visualize the fibrillization of amylin, a hormone which in aggregated forms plays a role in type 2 diabetes pathology. Data were obtained at acidic pH where fibrillization is hindered by the charging of histidine 18 and at slightly basic pH where the loss of charge on the histidine promotes aggregation. The experiments show three types of aggregate growth processes. In the earliest steps globular seeds are formed with some expanding radially during the course of the reaction. The dimensions of the globular seeds as well as their staining with the amyloid-specific dye thioflavin T indicate that they are plaques of short fibrils. The next species observed are fibrils that invariably grow from large globular seeds or smaller punctate granules. Fibril elongation appears to be unidirectional, although in some cases multiple fibrils radiate from a single seed or granule. After fibrils are formed, some show an increase in fluorescence intensity that we attribute to the growth of new fibrils alongside those previously formed. All three aggregation processes are suggestive of secondary (heterogeneous) nucleation mechanisms in which nucleation occurs on preformed fibrils. Consistently, electron micrographs show changes in fibril morphology well after fibrils are first formed, and the growth processes observed by fluorescence microscopy occur after the corresponding solution reactions have reached an initial apparent plateau. Taken together, the results highlight the importance of secondary nucleation in the fibrillization of amylin, as this could provide a pathway to continue fibril growth once an initial population of fibrils is established.     

Imaging the activity and localization of single voltage-gated Ca(2+) channels by total internal reflection fluorescence microscopy

The patch-clamp technique has enabled functional studies of single ion channels, but suffers limitations including lack of spatial information and inability to independently monitor currents from more than one channel. Here, we describe the use of total internal reflection fluorescence microscopy as an alternative, noninvasive approach to optically monitor the activity and localization of multiple Ca(2+)-permeable channels in the plasma membrane. Images of near-membrane Ca(2+) signals were obtained from >100 N-type channels expressed within restricted areas (80 x 80 micro m) of Xenopus oocytes, thereby permitting simultaneous resolution of their gating kinetics, voltage dependence, and localization. Moreover, this technique provided information inaccessible by electrophysiological means, demonstrating that N-type channels are immobile in the membrane, show a patchy distribution, and display diverse gating kinetics even among closely adjacent channels. Total internal reflection fluorescence microscopy holds great promise for single-channel recording of diverse voltage- and ligand-gated Ca(2+)-permeable channels in the membrane of neurons and other isolated or cultured cells, and has potential for high-throughput functional analysis of single channels.     

A novel subcellular collagen organization process visualized by total internal reflection fluorescence microscopy

The alpha1beta1 and alpha2beta1 integrins belong to a family of cell-surface molecules involved in structural contacts and signal-transduction events across the cell membrane. Employing two-dimensional substrates coated with fluorescently labeled type I collagen, we have discovered a novel subcellular matrix remodeling event that is particular to cells that express the fibrillar collagen receptor alpha2beta1. Cells expressing alpha1beta1 also perform this collagen organization process, but less proficiently. This work will provide a basis for subsequent studies of cell-mediated collagen fibril assembly.     

Two-dimensional standing wave total internal reflection fluorescence microscopy: superresolution imaging of single molecular and biological specimens

The development of high resolution, high speed imaging techniques allows the study of dynamical processes in biological systems. Lateral resolution improvement of up to a factor of 2 has been achieved using structured illumination. In a total internal reflection fluorescence microscope, an evanescence excitation field is formed as light is total internally reflected at an interface between a high and a low index medium. The <100 nm penetration depth of evanescence field ensures a thin excitation region resulting in low background fluorescence. We present even higher resolution wide-field biological imaging by use of standing wave total internal reflection fluorescence (SW-TIRF). Evanescent standing wave (SW) illumination is used to generate a sinusoidal high spatial frequency fringe pattern on specimen for lateral resolution enhancement. To prevent thermal drift of the SW, novel detection and estimation of the SW phase with real-time feedback control is devised for the stabilization and control of the fringe phase. SW-TIRF is a wide-field superresolution technique with resolution better than a fifth of emission wavelength or approximately 100 nm lateral resolution. We demonstrate the performance of the SW-TIRF microscopy using one- and two-directional SW illumination with a biological sample of cellular actin cytoskeleton of mouse fibroblast cells as well as single semiconductor nanocrystal molecules. The results confirm the superior resolution of SW-TIRF in addition to the merit of a high signal/background ratio from TIRF microscopy.     

Cell-substrate contacts illuminated by total internal reflection fluorescence

A technique for exciting fluorescence exclusively from regions of contact between cultured cells and the substrate is presented. The technique utilizes the evanescent wave of a totally internally reflecting laser beam to excite only those fluorescent molecules within one light wavelength or less of the substrate surface. Demonstrations of this technique are given for two types of cell cultures: rat primary myotubes with acetylcholine receptors labeled by fluorescent alpha- bungarotoxin and human skin fibroblasts labeled by a fluorescent lipid probe. Total internal reflection fluorescence examination of cells appears to have promising applications, including visualization of the membrane and underlying cytoplasmic structures at cell-substrate contacts, dramatic reduction of autofluorescence from debris and thick cells, mapping of membranes topography, and visualization of reversible bound fluorescent ligands at membrane receptors.     

Deconstructing the late phase of vimentin assembly by total internal reflection fluorescence microscopy (TIRFM)

Quantitative imaging of intermediate filaments (IF) during the advanced phase of the assembly process is technically difficult, since the structures are several μm long and therefore they exceed the field of view of many electron (EM) or atomic force microscopy (AFM) techniques. Thereby quantitative studies become extremely laborious and time-consuming. To overcome these difficulties, we prepared fluorescently labeled vimentin for visualization by total internal reflection fluorescence microscopy (TIRFM). In order to investigate if the labeling influences the assembly properties of the protein, we first determined the association state of unlabeled vimentin mixed with increasing amounts of labeled vimentin under low ionic conditions by analytical ultracentrifugation. We found that bona fide tetrameric complexes were formed even when half of the vimentin was labeled. Moreover, we demonstrate by quantitative atomic force microscopy and electron microscopy that the morphology and the assembly properties of filaments were not affected when the fraction of labeled vimentin was below 10%. Using fast frame rates we observed the rapid deposition of fluorescently labeled IFs on glass supports by TIRFM in real time. By tracing their contours, we have calculated the persistence length of long immobilized vimentin IFs to 1 μm, a value that is identical to those determined for shorter unlabeled vimentin. These results indicate that the structural properties of the filaments were not affected significantly by the dye. Furthermore, in order to analyze the late elongation phase, we mixed long filaments containing either Alexa 488- or Alexa 647-labeled vimentin. The 'patchy' structure of the filaments obtained unambiguously showed the elongation of long IFs through direct end-to-end annealing of individual filaments.     

Structural organization of interphase 3T3 fibroblasts studied by total internal reflection fluorescence microscopy

We studied the laminar organization of 3T3 fibroblast cells growing on glass slides by use of total internal reflection illumination to excite fluorescence emission (TIRF) from labeled molecules and stained cellular compartments that are very close to the cell-substrate contact region. Mitochondria, distant from the contact regions and stained with the water-soluble cationic dye, dil-C3-(3), fluoresced only as the glass/cytoplasm critical angle was approached. A similar result was obtained when the nuclei were stained with Hoechst dye 33342. From this measured angle a cytoplasmic refractive index in the range 1.358-1.374 was computed. The plasma membrane of 3T3 cells was stained with dil-C18-(3), and the cytoplasmic compartment was stained with fluoresceinyl-dextran (FTC-dextran) or with carboxyfluorescein. We have demonstrated a high degree of correspondence between the low-reflectance zones in the reflection interference image of a live cell and the TIRF images of both the plasma membrane and cytoplasmic compartment. TIRF photometry of selected contact regions of cells provided data from which the absolute separation of cell and substrate was computed. From a population of 3T3 cells microinjected with fluorescein-labeled actin, motile and adherent interphase cells were selected for study. For adherent cells, which displayed fluorescent stress fibers, the TIRF image was composed of intense patches and less intense regions that corresponded, respectively, to the focal contact and close-contact zones of the reflection-interference image. The intense patches corresponded to the endpoints of the stress fibers. Cells of motile morphology, which formed some focal contacts and extensive close-contact zones, gave AF-actin TIRF images of relatively even intensity. Thin lamellar regions of the cytoplasm were found to contain concentrations of actin not significantly different from other close-contact regions of the cell. The major analytical problem of TIRF microscopy is separation of the effects of proximity to substrate, refractive index, and fluorescent probe concentration on the local brightness of the TIRF image. From our results, it appears possible to use TIRF microscopy to measure the proximity of different components of substrate contact regions of cells.     

Endocytic adaptor molecules reveal an endosomal population of clathrin by total internal reflection fluorescence microscopy

Most eukaryotes utilize a single pool of clathrin to assemble clathrin-coated transport vesicles at different intracellular locations. Coat assembly is a cyclical process. Soluble clathrin triskelia are recruited to the membrane surface by compartment-specific adaptor and/or accessory proteins. Adjacent triskelia then pack together to assemble a polyhedral lattice that progressively invaginates, budding off the membrane surface encasing a nascent transport vesicle that is quickly uncoated. Using total internal reflection fluorescence microscopy to follow clathrin dynamics close to the cell surface, we find that the majority of labeled clathrin structures are relatively static, moving vertically in and out of the evanescent field but with little lateral motion. A small minority shows rapid lateral and directed movement over micrometer distances. Adaptor proteins, including the alpha subunit of AP-2, ARH, and Dab2 are also relatively static and exhibit virtually no lateral movement. A fluorescently labeled AP-2 beta2 subunit, incorporated into both AP-2 and AP-1 adaptor complexes, exhibits both types of behavior. This suggests that the highly motile clathrin puncta may be distinct from plasma membrane-associated clathrin structures. When endocytosed cargo molecules, such as transferrin or low density lipoprotein, are followed into cells, they exhibit even more lateral motion than clathrin, and gradually concentrate in the perinuclear region, consistent with classical endosomal trafficking. Importantly, clathrin partially colocalizes with internalized transferrin, but diverges as the structures move longitudinally. Thus, highly motile clathrin structures are apparently distinct from the plasma membrane, accompany transferrin, and contain AP-1, revealing an endosomal population of clathrin structures.     

Imaging single-channel calcium microdomains by total internal reflection microscopy

The microdomains of Ca2+ in the cytosol around the mouth of open Ca2+ channels are the basic 'building blocks' from which cellular Ca2+ signals are constructed. Moreover, the kinetics of local [Ca2+] closely reflect channel gating, so their measurement holds promise as an alternative to electrophysiological patch-clamp recording as a means to study single channel behavior. We have thus explored the development of optical techniques capable of imaging single-channel Ca2+ signals with good spatial and temporal resolution, and describe results obtained using total internal reflection fluorescence microscopy to monitor Ca2+ influx through single N-type channels expressed in Xenopus oocytes.     

New insights into the spliceosome by single molecule fluorescence microscopy

Splicing is an essential eukaryotic process in which introns are excised from precursors to messenger RNAs and exons ligated together. This reaction is catalyzed by a multi-MegaDalton machine called the spliceosome, composed of 5 small nuclear RNAs (snRNAs) and a core set of ～100 proteins minimally required for activity. Because of the spliceosome's size, its low abundance in cellular extracts, and its highly dynamic assembly pathway, analysis of the kinetics of splicing and the conformational rearrangements occurring during spliceosome assembly and disassembly has proven extraordinarily challenging. Here, we review recent progress in combining chemical biology methodologies with single molecule fluorescence techniques to provide a window into splicing in real time. These methods complement ensemble measurements of splicing in vivo and in vitro to facilitate kinetic dissection of pre-mRNA splicing.     

Elongation of amyloid fibrils through lateral binding of monomers revealed by total internal reflection fluorescence microscopy

Amyloid fibrils are fibrillar aggregates of denatured proteins associated with a large number of amyloidoses. The formation of amyloid fibrils has been considered to occur by nucleation and elongation. Real-time imaging of the elongation as well as linear morphology of amyloid fibrils suggests that all elongation events occur at the growing ends of fibrils. On the other hand, we suggested that monomers also bind to the lateral sides of preformed fibrils during the seed-dependent elongation, diffuse to the growing ends, and finally make further conformation changes to the mature amyloid fibrils. To examine lateral binding during the elongation of fibrils, we used islet amyloid polypeptide (IAPP), which has been associated with type II diabetes, and prepared IAPP modified with the fluorescence dye, Alexa532. By monitoring the elongation process with amyloid specific thioflavin T and Alexa532 fluorescence, we obtained overlapping images of the two fluorescence probes, which indicated lateral binding. These results are similar to the surface diffusion-dependent growth of crystals, further supporting the similarities between amyloid fibrillation and the crystallization of substances.     

Orientation distributions for cytochrome c on polar and nonpolar interfaces by total internal reflection fluorescence.

The formation of chemisorbed monolayers of yeast cytochrome c on both uncharged polar and nonpolar soft surfaces of organic self-assembled monolayers (SAM) on solid inorganic substrates was followed in situ by polarized total internal reflection fluorescence. Two types of nonpolar surfaces and one type of uncharged polar surface were used. The first type of nonpolar surface contained only thiol endgroups, while the other was composed of a mixture of thiol and methyl endgroups. The uncharged polar surface was provided by the mixture of thiol and hydroxyl endgroups. The thiol endgroups were used to form a covalent disulfide bond with the unique surface-exposed cysteine residue 102 of the protein. The mean tilt angle of the protein's zinc-substituted porphyrin was found to be 41 degrees and 50 degrees for the adsorption onto the nonpolar and uncharged polar surfaces, respectively. The distribution widths for the pure thiol and the thiol/methyl and thiol/hydroxyl mixtures were 9 degrees, 1 degrees, and 18 degrees, respectively. The high degree of the orientational order and good stability achieved for the protein monolayer on the mixed thiol/methyl endgroup SAM makes this system very attractive for studies of both intramolecular and intermolecular electron transfer processes.     

Binding of proteins to specific target sites in membranes measured by total internal reflection fluorescence microscopy

A new quantitative technique for measuring the binding of proteins to membranes is described. The method is based on a combination of total internal reflection fluorescence microscopy and the preparation of supported planar bilayers. Specific and reversible binding of a fluorescence-labeled monoclonal antibody to lipid haptens that were embedded in supported bilayers has been measured by this technique and compared to binding experiments that were conducted on membrane vesicles in solution. Equilibrium binding constants and kinetic parameters have been determined and used to expand the picture of the antibody-lipid hapten reaction. Estimates demonstrate that this technique is capable of measuring a broad range of binding constants (down to about 10(4) M-1) using only small amounts of ligand and receptor.