Super-Resolution Imaging in Fluid Mechanics Using New Illumination Approaches

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 (TIRFM) is an elegant optical technique that provides for the excitation of fluorophores in an extremely thin axial region ("optical section"). The method is based on the principle that when excitation light is totally internally reflected in a transparent solid (e.g., coverglass) at its interface with liquid, an electromagnetic field, called the evanescent wave, is generated in the liquid at the solid-liquid interface and is the same frequency as the excitation light. Since the intensity of the evanescent wave exponentially decays with distance from the surface of the solid, only fluorescent molecules within a few hundred nanometers of the solid are efficiently excited. This unit will briefly review the history, optical theory, and different hardware configurations used in TIRFM. In addition, it will provide experimental details and methodological considerations for studying receptors at the plasma membrane in neurons.

SDF 1-alpha (CXCL12) triggers glutamate exocytosis from astrocytes on a millisecond time scale: Imaging analysis at the single-vesicle level with TIRF microscopy

1] Total internal reflection fluorescence microscopy in cell biology

A major waste byproduct of oil sands in situ extraction is oil sands tailings, which are a mixture of water, clay, and residual bitumen. These tailings represent a huge ecological footprint in the form of tailings ponds, which not only render large land areas unusable but also prevent reuse of water. The slow dewatering of the tailings ponds poses a major challenge to the industry. The presence of complex inorganic–organic bitumen–clay mixtures in these tailings contributes to this problem. Hence, understanding the nature of the bitumen–clay association and the effect of bitumen on clay particle–particle interactions is important for the development of more effective chemicals or processes to accelerate particle aggregation and sedimentation during dewatering. Previous studies that investigate these interactions used techniques that are sensitive only toward inorganic clay but not sensitive towards organic bitumen. Here, we use a high-resolution total internal reflection fluorescence (TIRF) microscopy to ...

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

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.

The selective microscopic imaging of the plasma membrane and adjacent structures by total internal reflection fluorescence (TIRF) microscopy is a versatile and frequently used technique in cell biology. A reduction of imaging artifacts in objective-type TIRF microscopy can be achieved by circular or multi-spot laser illumination or by using noncoherent light sources that are projected into the back focal plane as a light annulus. Light-emitting diode (LED)-based TIRF excitation is a recent advancement of the latter strategy. While some basic principles of LED-TIRF remain the same as in laser-based methods, the calculation of penetration depth, the flatness of illumination and the amount of available illumination power differ. This study provides the theoretical framework for the construction and adjustment of LED-TIRF. Using state-of-the art high power LED emitters, LED-TIRF achieves excitation efficiencies that are comparable to laser-based systems and homogenously illuminate the entire field of view, thus, allowing variation of the penetration depth or quantitative photobleaching-assisted imaging protocols. Using autofluorescent transmembrane, soluble and membrane-attached fusion proteins, we provide examples for a photobleaching-based assessment of the exchange kinetics of proteins within living human endothelial cells.

Alexa Fluor 647 is a widely used fluorescent probe for cell bioimaging and super-resolution microscopy. Herein, the reversible fluorescence switching of Alexa Fluor 647 conjugated to bovine serum albumin (BSA) and adsorbed onto indium tin oxide (ITO) electrodes under electrochemical potential control at the level of single protein molecules is reported. The modulation of the fluorescence as a function of potential was observed using total internal reflectance fluorescence (TIRF) microscopy. The fluorescence intensity of the Alexa Fluor 647 decreased, or reached background levels, at reducing potentials but returned to normal levels at oxidizing potentials. These electrochemically induced changes in fluorescence were sensitive to pH despite that BSA-Alexa Fluor 647 fluorescence without applied potential is insensitive to pH between values of 4-10. The observed pH dependence indicated the involvement of electron and proton transfer in the fluorescence switching mechanism.

Contributions of the lower dimer to supramolecular actin patterning revealed by TIRF microscopy.

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.

Kinesin-5 s are bipolar motor proteins that contribute to cell division by crosslinking and sliding apart antiparallel microtubules inside the mitotic spindle. However, the mechanism underlying the interactions between kinesin-5 and the microtubule remains poorly understood. In this study, we investigated the binding of BimC, a kinesin-5 motor from Aspergillus nidulans, to the microtubule using a combination of total internal reflection fluorescence (TIRF) microscopy and molecular dynamics (MD) simulations. TIRF microscopy experiments revealed that increasing the concentration of KCl in the motility buffer from 0 mM to 150 mM completely abolishes the ability of BimC to bind to the microtubule. Consistent with this experimental finding, MD simulations demonstrated a significant reduction in the strength of electrostatic interactions between BimC and microtubules at 150 mM KCl compared to 0 mM KCl. Furthermore, we identified several salt bridges at the BimC-microtubule interface, with positively charged residues on BimC interacting with negatively charged residues on the tubulin heterodimer. These results provide mechanistic insights into the role of electrostatic interactions in kinesin-5–microtubule binding, advancing our understanding of the molecular underpinnings of kinesin-5 motility.

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.

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.

This paper investigates plasmonic amplification in two commonly used optical configurations for fluorescence readout of bioassays – epifluorescence (EPF) and total internal reflection fluorescence (TIRF). The plasmonic amplification in the EPF configuration was implemented by using crossed gold diffraction grating and Kretschmann geometry of attenuated total reflection method (ATR) was employed in the TIRF configuration. Identical assay, surface architecture for analyte capture, and optics for the excitation, collection and detection of emitted fluorescence light intensity were used in both TIRF and EPF configurations. Simulations predict that the crossed gold diffraction grating (EPF) can amplify the fluorescence signal by a factor of 102 by the combination of surface plasmon-enhanced excitation and directional surface plasmon-coupled emission in the red part of spectrum. This factor is about order of magnitude higher than that predicted for the Kretschmann geometry (TIRF) which only took advantage of the surface plasmon-enhanced excitation. When applied for the readout of sandwich interleukin 6 (IL-6) immunoassay, the plasmonically amplified EPF geometry designed for Alexa Fluor 647 labels offered 4-times higher fluorescence signal intensity compared to TIRF. Interestingly, both geometries allowed reaching the same detection limit of 0.4pM despite of the difference in the fluorescence signal enhancement. This is attributed to inherently lower background of fluorescence signal for TIRF geometry compared to that for EPF which compensates for the weaker fluorescence signal enhancement. The analysis of the inflammation biomarker IL-6 in serum at medically relevant concentrations and the utilization of plasmonic amplification for the fluorescence measurement of kinetics of surface affinity reactions are demonstrated for both EPF and TIRF readout.