High refractive index silicone gels for simultaneous total internal reflection fluorescence and traction force microscopy of adherent cells

Substrate rigidity profoundly impacts cellular behaviors such as migration, gene expression, and cell fate. Total Internal Reflection Fluorescence (TIRF) microscopy enables selective visualization of the dynamics of substrate adhesions, vesicle trafficking, and biochemical signaling at the cell-substrate interface. Here we apply high-refractive-index silicone gels to perform TIRF microscopy on substrates with a wide range of physiological elastic moduli and simultaneously measure traction forces exerted by cells on the substrate.     

Spatially and temporally synchronized atomic force and total internal reflection fluorescence microscopy for imaging and manipulating cells and biomolecules

The atomic force microscope is a high-resolution scanning-probe instrument which has become an important tool for cellular and molecular biophysics in recent years but lacks the time resolution and functional specificities offered by fluorescence microscopic techniques. To exploit the advantages of both methods, here we developed a spatially and temporally synchronized total internal reflection fluorescence and atomic force microscope system. The instrument, which we hereby call STIRF-AFM, is a stage-scanning device in which the mechanical and optical axes are coaligned to achieve spatial synchrony. At each point of the scan the sample topography (atomic force microscope) and fluorescence (photon count or intensity) information are simultaneously recorded. The tool was tested and validated on various cellular (monolayer cells in which actin filaments and intermediate filaments were fluorescently labeled) and biomolecular (actin filaments and titin molecules) systems. We demonstrate that with the technique, correlated sample topography and fluorescence images can be recorded, soft biomolecular systems can be mechanically manipulated in a targeted fashion, and the fluorescence of mechanically stretched titin can be followed with high temporal resolution.     

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.     

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.     

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.     

Total internal reflection fluorescence microscopy for single-molecule imaging in living cells

Marvelous background rejection in total internal reflection fluorescence microscopy (TIR-FM) has made it possible to visualize single-fluorophores in living cells. Cell signaling proteins including peptide hormones, membrane receptors, small G proteins, cytoplasmic kinases as well as small signaling compounds have been conjugated with single chemical fluorophore or tagged with green fluorescent proteins and visualized in living cells. In this review, the reasons why single-molecule analysis is essential for studies of intracellular protein systems such as cell signaling system are discussed, the instrumentation of TIR-FM for single-molecule imaging in living cells is explained, and how single molecule visualization has been used in cell biology is illustrated by way of two examples: signaling of epidermal growth factor in mammalian cells and chemotaxis of Dictyostelium amoeba along a cAMP gradient. Single-molecule analysis is an ideal method to quantify the parameters of reaction dynamics and kinetics of unitary processes within intracellular protein systems. Knowledge of these parameters is crucial for the understanding of the molecular mechanisms underlying intracellular events, thus single-molecule imaging in living cells will be one of the major technologies in cellular nanobiology.     

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.     

Quantifying exocytosis by combination of membrane capacitance measurements and total internal reflection fluorescence microscopy in chromaffin cells

Total internal reflection fluorescence microscopy (TIRF-Microscopy) allows the observation of individual secretory vesicles in real-time during exocytosis. In contrast to electrophysiological methods, such as membrane capacitance recording or carbon fiber amperometry, TIRF-Microscopy also enables the observation of vesicles as they reside close to the plasma membrane prior to fusion. However, TIRF-Microscopy is limited to the visualization of vesicles that are located near the membrane attached to the glass coverslip on which the cell grows. This has raised concerns as to whether exocytosis measured with TIRF-Microscopy is comparable to global secretion of the cell measured with membrane capacitance recording. Here we address this concern by combining TIRF-Microscopy and membrane capacitance recording to quantify exocytosis from adrenal chromaffin cells. We found that secretion measured with TIRF-Microscopy is representative of the overall secretion of the cells, thereby validating for the first time the TIRF method as a measure of secretion. Furthermore, the combination of these two techniques provides a new tool for investigating the molecular mechanism of synaptic transmission with combined electrophysiological and imaging techniques.     

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.     

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.     

Reversible and irreversible adhesion of motile Escherichia coli cells analyzed by total internal reflection aqueous fluorescence microscopy

The initial events in bacterial adhesion are often explained as resulting from electrostatic and van der Waals forces between the cell and the surface, as described by DLVO theory (developed by Derjaguin, Landau, Verwey, and Overbeek). Such a theory predicts that negatively charged bacteria will experience greater attraction toward a negatively charged surface as the ionic strength of the medium is increased. In the present study we observed both smooth-swimming and nonmotile Escherichia coli bacteria close to plain, positively, and hydrophobically coated quartz surfaces in high- and low-ionic-strength media by using total internal reflection aqueous fluorescence microscopy. We found that reversibly adhering cells (cells which continue to swim along the surface for extended periods) are too distant from the surface for this behavior to be explained by DLVO-type forces. However, cells which had become immobilized on the surface did seem to be affected by electrostatic interactions. We propose that the "force" holding swimming cells near the surface is actually the result of a hydrodynamic effect, causing the cells to swim at an angle along the glass, and that DLVO-type forces are responsible only for the observed immobilization of irreversibly adhering cells. We explain our observations within the context of a conceptual model in which bacteria that are interacting with the surface may be thought of as occupying one of three compartments: bulk fluid, near-surface bulk, and near-surface constrained. A cell in these compartments feels either no effect of the surface, only the hydrodynamic effect of the surface, or both the hydrodynamic and the physicochemical effects of the surface, respectively.     

Atomic force microscopy and chemical force microscopy of microbial cells

Over the past years, atomic force microscopy (AFM) has emerged as a powerful tool for imaging the surface of microbial cells with nanometer resolution, and under physiological conditions. Moreover, chemical force microscopy (CFM) and single-molecule force spectroscopy have enabled researchers to map chemical groups and receptors on cell surfaces, providing valuable insight into their structure-function relationships. Here, we present protocols for analyzing spores of the pathogen Aspergillus fumigatus using real-time AFM imaging and CFM. We emphasize the use of porous polymer membranes for immobilizing single live cells, and the modification of gold-coated tips with alkanethiols for CFM measurements. We also discuss recording conditions and data interpretation, and provide recommendations for reliable experiments. For well-trained AFM users, the entire protocol can be completed in 2-3 d.     

Visualization of cortex organization and dynamics in microorganisms, using total internal reflection fluorescence microscopy

TIRF microscopy has emerged as a powerful imaging technology to study spatio-temporal dynamics of fluorescent molecules in vitro and in living cells. The optical phenomenon of total internal reflection occurs when light passes from a medium with high refractive index into a medium with low refractive index at an angle larger than a characteristic critical angle (i.e. closer to being parallel with the boundary). Although all light is reflected back under such conditions, an evanescent wave is created that propagates across and along the boundary, which decays exponentially with distance, and only penetrates sample areas that are 100-200 nm near the interface. In addition to providing superior axial resolution, the reduced excitation of out of focus fluorophores creates a very high signal to noise ratios and minimizes damaging effects of photobleaching. Being a widefield technique, TIRF also allows faster image acquisition than most scanning based confocal setups. At first glance, the low penetration depth of TIRF seems to be incompatible with imaging of bacterial and fungal cells, which are often surrounded by thick cell walls. On the contrary, we have found that the cell walls of yeast and bacterial cells actually improve the usability of TIRF and increase the range of observable structures. Many cellular processes can therefore be directly accessed by TIRF in small, single-cell microorganisms, which often offer powerful genetic manipulation techniques. This allows us to perform in vivo biochemistry experiments, where kinetics of protein interactions and activities can be directly assessed in living cells. We describe here the individual steps required to obtain high quality TIRF images for Saccharomyces cerevisiae or Bacillus subtilis cells. We point out various problems that can affect TIRF visualization of fluorescent probes in cells and illustrate the procedure with several application examples. Finally, we demonstrate how TIRF images can be further improved using established image restoration techniques.     

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.     

Simultaneous multicolor detection system of the single-molecular microbial antigen with total internal reflection fluorescence microscopy

Immunological diagnostic methods have been widely performed and showed high performance in molecular and cellular biology, molecular imaging, and medical diagnostics. We have developed novel methods for the fluorescent labeling of several antibodies coupled with fluorescent nanocrystal QDs. In this study we demonstrated that two bacterial toxins, diphtheria toxin and tetanus toxin, were detected simultaneously in the same view field of a cover slip by using directly QD-conjugated antibodies. We have succeeded in detecting bacterial toxins by counting luminescent spots on the evanescent field with using primary antibody conjugated to QDs. In addition, each bacterial toxin in the mixture can be separately detected by single excitation laser with emission band pass filters, and simultaneously in situ pathogen quantification was performed by calculating the luminescent density on the surface of the cover slip. Our results demonstrate that total internal reflection fluorescence microscopy (TIRFM) enables us to distinguish each antigen from mixed samples and can simultaneously quantitate multiple antigens by QD-conjugated antibodies . Bioconjugated QDs could have great potentialities for in practical biomedical applications to develop various high-sensitivity detection systems.     

Atomic force microscopy for the study of membrane proteins

Fundamental biological processes such as cell-cell communication, signal transduction, molecular transport and energy conversion are performed by membrane proteins. These important proteins are studied best in their native environment, the lipid bilayer. The atomic force microscope (AFM) is the instrument of choice to determine the native surface structure, supramolecular organization, conformational changes and dynamics of membrane-embedded proteins under near-physiological conditions. In addition, membrane proteins are imaged at subnanometer resolution and at the single molecule level with the AFM. This review highlights the major advances and results achieved on reconstituted membrane proteins and native membranes as well as the recent developments of the AFM for imaging.