Total Internal Reflection Fluorescence (TIRF) Microscopy

Total internal reflection fluorescence (TIRF) microscopy has gained popularity in recent years among cell biologists due to its ability to clearly visualize events that occur at the adherent plasma membrane of cells. TIRF microscopy systems are now commercially available from nearly all microscope suppliers. This review aims to give the reader an introduction to the physical basis of TIRF and considerations that need to be made when purchasing a commercial system. We explain how TIRF can be combined with other microscopy modalities and describe how to use TIRF to study processes such as endocytosis, exocytosis, and focal adhesion dynamics. Finally, we provide a step-by-step guide to imaging and analyzing focal adhesion dynamics in a migrating cell using TIRF microscopy.

Total internal reflection fluorescence (TIRF) microscopy, used in conjunction with flash photolysis, provides a useful way of investigating the kinetics of macromolecular interactions. We compare different TIRF optical geometries to establish an optimal combination. Excitation light was introduced via four different arrangements: (1) a prism positioned on the microscope optical axis, (2) an offset prism with propagation through a silica slide trans to the objective lens, (3) an offset prism with propagation through a silica coverslip cis to a water-immersion objective lens and (4) a prismless arrangement using a high NA oil-immersion objective lens. Photolysis was achieved using a Xe flash lamp and a customised silica condenser lens. Single myosin molecules labelled with a Cy3 fluorophore were used as a test sample. Although the offset trans prism gave the best signal-to-background ratio, a customised thin rhombic prism incorporated, on axis, into the flash condenser assembly was almost as good and was more practical for scanning multiple fields. An oil-immersion lens gave the brightest image for sample depths < 30 micrometer but above this limit, a water-immersion lens was better. The prismless arrangement may offer advantages in other situations but it is important to check the actual numerical aperture of the objective lens.

Eliminating Unwanted Far-Field Excitation in Objective-Type TIRF. Part II. Combined Evanescent-Wave Excitation and Supercritical-Angle Fluorescence Detection Improves Optical Sectioning

In Alzheimer disease, amyloid beta, a 39-43-residue peptide produced by cleavage from a large amyloid precursor protein, undergoes conformational change to form amyloid fibrils and deposits as senile amyloid plaques in the extracellular cerebral cortices of the brain. However, the mechanism of how the intrinsically linear amyloid fibrils form spherical senile plaques is unknown. With total internal reflection fluorescence microscopy combined with the use of thioflavin T, an amyloid-specific fluorescence dye, we succeeded in observing the formation of the senile plaque-like spherulitic structures with diameters of around 15 microm on the chemically modified quartz surface. Real-time observation at a single fibrillar level revealed that, in the absence of tight contact with the surface, the cooperative and radial growth of amyloid fibrils from the core leads to a huge spherulitic structure. The results suggest the underlying physicochemical mechanism of senile plaque formation, essential for obtaining insight into prevention of Alzheimer disease.

Single molecule detection and fluorescence correlation spectroscopy on surfaces

TIRF Microscopy Studies of Axial Protein Organization using Photoswitchable Fluorescent Proteins

The brain contains glial cells. Astrocytes, a type of glial cell, have long been known to provide a passive supportive role to neurons. However, increasing evidence suggests that astrocytes may also actively participate in brain function through functional interactions with neurons. However, many fundamental aspects of astrocyte biology remain controversial, unclear and/or experimentally unexplored. One important issue is the dynamics of intracellular calcium transients in astrocytes. This is relevant because calcium is well established as an important second messenger and because it has been proposed that astrocyte calcium elevations can trigger the release of transmitters from astrocytes. However, there has not been any detailed or satisfying description of near plasma membrane calcium signaling in astrocytes. Total internal reflection fluorescence (TIRF) microscopy is a powerful tool to analyze physiologically relevant signaling events within about 100 nm of the plasma membrane of live cells. Here, we use TIRF microscopy and describe how to monitor near plasma membrane and global intracellular calcium dynamics almost simultaneously. The further refinement and systematic application of this approach has the potential to inform about the precise details of astrocyte calcium signaling. A detailed understanding of astrocyte calcium dynamics may provide a basis to understand if, how, when and why astrocytes and neurons undergo calcium-dependent functional interactions.

Imaging GPCRs trafficking and signaling with total internal reflection fluorescence microscopy in cultured neurons.

Total internal reflectance fluorescence (TIRF) microscopy is a technique that allows the study of events happening at the cell membrane, by selective imaging of fluorescent molecules that are closest to a high refractive index substance such as glass1. In this article, we apply this technique to image exocytosis of synaptic vesicles in retinal bipolar cells isolated from the goldfish retina. These neurons are very suitable for this kind of study due to their large axon terminals. By simultaneously patch clamping the bipolar cells, it is possible to investigate the relationship between pre-synaptic voltage and synaptic release2,3. Synaptic vesicles inside the bipolar cell terminals are loaded with a fluorescent dye (FM 1-43®) by co-puffing the dye and a ringer solution containing a high K+ concentration onto the synaptic terminals. This depolarizes the cells and stimulates endocytosis and consequent dye uptake into the glutamatergic vesicles. After washing the excess dye away for around 30 minutes, cells are ready for being patch clamped and imaged simultaneously with a 488 nm laser. The patch pipette solution contains a rhodamine-based peptide that binds selectively to the synaptic ribbon protein RIBEYE4, thereby labeling ribbons specifically when terminals are imaged with a 561 nm laser. This allows the precise localization of active zones and the separation of synaptic from extra-synaptic events.

In a total internal reflection fluorescence microscope, there is a 3D anisotropy in intensity of the excitation light. Using a mirror in a prism-based total internal reflection fluorescence microscope, we propose a very low-cost method to decrease the anisotropic property of the excitation light. Theoretically, we obtained the electric field of the transmitted light inside the sample as a function of the rotational angle of the mirror, polarization degree of the incident beam, and the refractive indices of the prism, immersion oil, cover glass, and sample. By using the refractive indices that are commercially available, proper practical combinations of the angle of mirror and polarization degree of the incident light are suggested to provide a merely 2D isotropic excitation light at the sample plane of a total internal reflection fluorescence microscope.

This study introduces total internal reflection fluorescence (TIRF) as a method for investigating the dynamics of a single laser-induced cavitation bubble in water within a thin layer near a solid surface. TIRF, supported by a planar waveguide, was employed to image cavitation bubble collapses near the liquid–solid interface at 50 thousand frames per second (kfps). Simultaneously, traditional side-view background illumination at 150 kfps captured the bubble dynamics. Dilution series and thickness estimation were conducted to determine interfering fluorescence signal contributions. The results confirm that most fluorescence is excited by the evanescent field rather than surface scattering or bulk scattering. The lifecycle of cavitation bubbles with moderate non-dimensional stand-off distances, γ = 1.15, 1.35, and 1.6, are discussed. We find that a thin liquid film persists between the bubble and the solid surface throughout the entire collapse process, except for randomly distributed micron-sized bubbles attached to the surface between the first and second collapse. Shock waves were captured with TIRF during the second collapse, centered around regions of the toroidal cavity exhibiting locally stronger dynamics. These regions, termed gas-filled strong collapse areas (SCAs), were also where microcracks formed during the second collapse of a single cavitation bubble. While not all SCAs resulted in microcracks, every observed microcrack could be traced back to a preceding SCA. To our knowledge, this is the first application of TIRF to observe cavitation bubble collapses with sub-millisecond time resolution.

1] Total internal reflection fluorescence microscopy in cell biology

Total internal reflection fluorescence microscopy (TIRF-M) provides low-invasive high-contrast surface imaging with optical sectioning of typically 100-200nm. Thus, TIRF-M has become an established tool for imaging surfaces, including cell membranes. For TIRF-M, a homogenous evanescent field of excitation over the whole field of view is generally desired for quantitative microscopy; however, this is not necessarily straightforward to generate with Gaussian beams. In recent years, several improvements on TIRF-M have been developed that have addressed non-uniform scattering fringes and other artifacts. Here, we introduce a cost-effective TIRF setup with a very low degree of complexity and no moving parts, which provides a flattop-like excitation profile. The setup uses a tunable laser ring zoom focus system to generate a full 360° TIRF illumination. Two axicon lenses and one focus lens allow for generation and control of the ring diameter to tune the TIRF excitation angle. We show that 360° laser illumination in combination with a radial polarizer will generate an evanescent Bessel-beam excitation field that exhibits a flattop intensity over an extended part of the field of view, and demonstrate the advantages of this axicon-based Bessel beam illumination for live-cell imaging.

The last stage of the constitutive secretory pathway is the delivery and fusion of post-Golgi transport vesicles (carriers) to the plasma membrane. However, it is still unclear where at the plasma membrane these carriers fuse, in a living cell. In this work we used total internal reflection fluorescence microscopy (TIR-FM) to study the delivery and fusion of post-Golgi carriers, as well as of recycling vesicles from the endosomal compartment, to the cell surface in live cells. TIR-FM is suited to image fluorescent molecules near the cell-substrate interphase, since it selectively illuminates the contact surface of cells. One aim of this work was to establish a quantitative method for the microscopic detection of single exocytic fusion events. Following the localization of fusion we answered basic, previously unresolved, questions in the field of membrane traffic. 1\. Imaging fusion of single post-Golgi carriers. A TIR-FM system was optimized to image exocytosis of single post- Golgi carriers. By labeling the carriers with a GFP-tagged membrane protein (vesicular stomatitis virus glycoprotein, VSVG), quantitative criteria for the microscopic detection of single carrier fusion events were established for the first time. Quantitative analysis of time-lapse images could clearly distinguish fusion of the carriers from both movement of carriers relative to the plasma membrane as well as lysis of carriers. The flattening of the carriers into the plasma membrane as well as the subsequent diffusion of the membrane cargo into the plasma membrane was resolved. The duration of the flattening process was found to depend on the size of the carrier, distinguishing small spherical from large tubular carriers. 2\. Role of microtubules in post-Golgi traffic of fibroblasts. The simul-taneous imaging of post-Golgi carriers and microtubules using a novel dual-color TIR-FM system showed that post-Golgi carriers are transported along microtubules to the fusion sites at the plasma membrane. The data strongly suggested that the carriers are capable of undergoing fusion while still attached to the microtubules and that the carriers do not have to reach the end of the microtubules in order to fuse. In contrast to stationary fibroblasts, migrating fibroblasts were shown to have a microtubule-mediated mechanism for polarized insertion of post-Golgi carriers (here LDL-receptor-GFP) close to the leading edge. Disrupting the microtubules restricted this directed delivery of the carriers to regions of the plasma membrane close to the Golgi complex, making the distribution of fusion sites in stationary and migrating cells indistinguishable. Disrupting the microtubules also decreased the overall fusion frequency, increased the frequency of "partial" fusions, and increased the amount of cargo delivered per fusion. We conclude that the microtubule cytoskeleton is necessary for the domain-specific delivery of post-Golgi membrane cargo in fibroblasts. 3\. Role of microtubules in post- Golgi traffic of polarized epithelial cells. Time-lapse fluorescence microscopy was used to analyze the delivery of apical and basolateral membrane proteins to the cell surface in both non-polarized and polarized epithelial cells. We demonstrated that post-Golgi carriers containing either apical or basolateral membrane proteins fuse to the basal membrane in non-polarized cells. Upon polarization, exocytosis of all carriers to the basal membrane was abrogated. Basolateral carriers were seen to fuse to sites at the lateral membrane, while apical carriers presumably fused to the apical membrane. This selective targeting is concomitant with redistribution of the t-SNAREs, syntaxin 3 and 4, upon polarization. Furthermore, we showed that both the targeted exocytosis of apical proteins and the exclusive localization of syntaxin 3 at the apical plasma membrane are dependent on intact microtubules in polarized epithelial cells. In contrast, targeted exocytosis of basolateral proteins and the basolateral distribution of syntaxin 4 and sec6 are maintained independently of microtubules in polarized cells. 4\. Insulin- regulated recycling of glucose transporter. We studied the insulin-regulated release of the glucose transporter (GLUT4) from the endosomal recycling compartment (ERC) in live cells. We show that GLUT4 is retained within the transferrin receptor-containing general ERC in fibroblasts. Using dual-color TIR-FM, we demonstrate that the transferrin receptor and GLUT4 are transported from the ERC in separate vesicles. This provides the first functional evidence for the formation of distinct classes of vesicles from the ERC. We propose that GLUT4 is dynamically retained within the ERC in fibroblasts because it is concentrated in vesicles that form more slowly than those that transport transferrin receptor.

SummaryFluidity is essential for many biological membrane functions. The basis for understanding membrane structure remains the classic Singer-Nicolson model, in which proteins are embedded within a fluid lipid bilayer and able to diffuse laterally within a sea of lipid. Here we report lipid and protein diffusion in the plasma membrane of live cells of the bacterium Escherichia coli, using Fluorescence Recovery after Photobleaching (FRAP) and Total Internal Reflection Fluorescence (TIRF) microscopy to measure lateral diffusion coefficients. Lipid and protein mobility within the membrane were probed by visualizing an artificial fluorescent lipid and a simple model membrane protein consisting of a single membrane-spanning alpha-helix with a Green Fluorescent Protein (GFP) tag on the cytoplasmic side. The effective viscosity of the lipid bilayer is strongly temperature-dependent, as indicated by changes in the lipid diffusion coefficient. Surprisingly, the mobility of the model protein was unaffected by changes in the effective viscosity of the bulk lipid, and TIRF microscopy indicates that it clusters in segregated, mobile domains. We suggest that this segregation profoundly influences the physical behaviour of the protein in the membrane, with strong implications for bacterial membrane function and bacterial physiology.