Total Internal Reflection Fluorescence Research Articles

Modules for teaching and applying single-molecule total internal reflection fluorescence microscopy in an undergraduate lab course

Modules for teaching and applying single-molecule total internal reflection fluorescence microscopy in an undergraduate lab course

Quantifying tau-microtubule interactions with TIRF microscopy and FCS

Quantifying tau-microtubule interactions with TIRF microscopy and FCS

Antibody surface mobility amplifies FcγR signaling via Arp2/3 during phagocytosis

We report herein that the anti-CD20 therapeutic antibody, rituximab, is rearranged into microclusters within the phagocytic synapse by macrophage Fcγ receptors (FcγR) during antibody-dependent cellular phagocytosis. These microclusters were observed to potently recruit Syk and to undergo rearrangements that were limited by the cytoskeleton of the target cell, with depolymerization of target-cell actin filaments leading to modest increases in phagocytic efficiency. Total internal reflection fluorescence analysis revealed that FcγR total phosphorylation, Syk phosphorylation, and Syk recruitment were enhanced when IgG-FcγR microclustering was enabled on fluid bilayers relative to immobile bilayers in a process that required Arp2/3. We conclude that on fluid surfaces, IgG-FcγR microclustering promotes signaling through Syk that is amplified by Arp2/3-driven actin rearrangements. Thus, the surface mobility of antigens bound by IgG shapes the signaling of FcγR with an unrecognized complexity beyond the zipper and trigger models of phagocytosis.

CAR-mediated targeting of NK cells overcomes tumor immune escape caused by ICAM-1 downregulation

BackgroundThe antitumor activity of natural killer (NK) cells can be enhanced by specific targeting with therapeutic antibodies that trigger antibody-dependent cell-mediated cytotoxicity (ADCC) or by genetic engineering to express chimeric...

Disruption Mechanisms of Enveloped Viruses by Ionic and Nonionic Surfactants.

The world has witnessed multiple pandemics and endemics caused by enveloped viruses in the past century. To name a few, the ongoing COVID-19 pandemic and other pandemics/endemics caused by coronaviruses, influenza viruses, HIV-1, etc. The external and topical applications of surfactants have been effective in limiting the spread of viruses. While it is well-known that surfactants inactivate virus particles (virions), the mechanism of action of surfactants against enveloped virions has not yet been established. In this work, we have evaluated the surfactant-induced disruption mechanism of a cocktail of enveloped viruses containing particles of mumps, measles, and rubella viruses. We applied the total internal reflection fluorescence microscopy technique to trace the temporal changes in the fluorescence signal from single virions upon the addition of a surfactant solution. We report that surfactants solubilize either the viral lipid membrane, proteins, or both. Ionic surfactants, depending on their charge and interaction type with the viral lipids and proteins, can cause bursting or perforation of the viral envelope, whereas a nonionic surfactant can cause either symmetric expansion or perforation of the viral envelope depending on the surfactant concentration.

All SNAP25 molecules in the vesicle-plasma membrane contact zone change conformation during vesicle priming.

In neuronal cell types, vesicular exocytosis is governed by the SNARE (soluble NSF attachment receptor) complex consisting of synaptobrevin2, SNAP25, and syntaxin1. These proteins are required for vesicle priming and fusion. We generated an improved SNAP25-based SNARE COmplex Reporter (SCORE2) incorporating mCeruelan3 and Venus and overexpressed it in SNAP25 knockout embryonic mouse chromaffin cells. This construct rescues vesicle fusion with properties indistinguishable from fusion in wild-type cells. Combining electrochemical imaging of individual release events using electrochemical detector arrays with total internal reflection fluorescence resonance energy transfer (TIR-FRET) imaging reveals a rapid FRET increase preceding individual fusion events by 65 ms. The experiments are performed under conditions of a steady-state cycle of docking, priming, and fusion, and the delay suggests that the FRET change reflects tight docking and priming of the vesicle, followed by fusion after ~65 ms. Given the absence of wt SNAP25, SCORE2 allows determination of the number of molecules at fusion sites and the number that changes conformation. The number of SNAP25 molecules changing conformation in the priming step increases with vesicle size and SNAP25 density in the plasma membrane and equals the number of copies present in the vesicle-plasma membrane contact zone. We estimate that in wt cells, 6 to 7 copies of SNAP25 change conformation during the priming step.

Biohybrid Photonic Platform for Subcellular Stimulation and Readout of In Vitro Neurons.

Targeted manipulation of neural activity via light has become an indispensable tool for gaining insights into the intricate processes governing single neurons and complex neural networks. To shed light onto the underlying interaction mechanisms, it is crucial to achieve precise control of individual neural activity, as well as a spatial read-out resolution on the nanoscale. Here, a versatile photonic platform with subcellular resolution for stimulation and monitoring of in-vitro neurons is demonstrated. Low-loss photonic waveguides are fabricated on glass substrates using nanoimprint lithography and featuring a loss of only -0.9±0.2dBcm-1 at 489nm and are combined with optical fiber-based waveguide-access and backside total internal reflection fluorescence microscopy. Neurons are grown on the bio-functionalized photonic chip surface and, expressing the light-sensitive ion channel Channelrhodopsin-2, are stimulated within the evanescent field penetration depth of 57nm of the biocompatible waveguides. The versatility and cost-efficiency of the platform, along with the possible subcellular resolution, enable tailor-made investigations of neural interaction dynamics with defined spatial control and high throughput.

Bacterial surface swimming states revealed by TIRF microscopy.

Motility near solid surfaces plays a key role in the life cycle of bacteria and is essential for biofilm formation, biofilm dispersal, and virulence. The alignment of the cell body with the surface during surface swimming impacts bacterial surface sensing. Here, we developed a high-throughput method for characterizing the orientation of the cell body relative to the surface using total internal reflection fluorescence (TIRF) microscopy. The angle between the cell body and the surface was determined by maximizing image cross-correlations between the TIRF image of the cell and a reference library. Utilizing this technique, we surprisingly identified six distinct surface swimming states of Pseudomonas aeruginosa according to the body alignment and the flagellar position. Furthermore, we observed that the near-surface swimming speed is greater in the pull state than in the push state, attributed to hydrodynamic effects near the liquid-solid interface. Hydrodynamic force analysis of the swimming states provided rich insights into the mechanics of bacterial surface swimming. Our technique is readily applicable to the study of surface motility across a wide spectrum of bacterial species.

Injectable extracellular matrix-mimetic hydrogel based on electrospun Janus fibers.

To date, the reported injectable hydrogels have failed to mimic the fibrous architecture of the extracellular matrix (ECM), limiting their biological effects on cell growth and phenotype. Additionally, they lack the micro-sized pores present within the ECM, which is unfavorable for the facile transport of nutrients and waste. Herein, an injectable ECM-mimetic hydrogel (IEMH) was fabricated by shortening and dispersing Janus fibers capable of self-curling at body temperature into pH 7.4 phosphate buffer solution. The IEMH could be massively prepared through a side-by-side electrospinning process combined with ultraviolet irradiation. The IEMHs with only 5 wt% fibers could undergo sol-gel transition at body temperature to become solid gels with desirable stability, sturdiness, and elasticity and self-healing ability. In addition, they possessed notable pseudoplasticity, which is beneficial to injection at room temperature. The results obtained from characterization analysis via scanning electron microscopy, total internal reflection fluorescence microscopy, nuclear magnetic resonance spectroscopy, and Fourier-transform infrared spectroscopy indicate that their sol-gel transition under physiological conditions stems from the synergistic action of the tight entanglements between thermally-induced self-curling fibers and the hydrophobic interaction between the fibers. An MTT assay using C2C12 myoblast cells was performed to examine the in vitro cytotoxicity of IEMHs for biomedical applications, and the cell viability was found to be more than 95%.

Practical applications of total internal reflection fluorescence microscopy for nanocatalysis

A ‘pocket guide’ to applications of total internal reflection fluorescence in the field of chemistry.

Visualizing HIV-1 Assembly at the T-Cell Plasma Membrane Using Single-Molecule Localization Microscopy.

The 20-year revolution in optical fluorescence microscopy, supported by the optimization of both spatial resolution and timely acquisition, allows the visualization of nanoscaled objects in cell biology. Currently, the use of a recent generation of super-resolution fluorescence microscope coupled with improved fluorescent probes gives the possibility to study the replicative cycle of viruses in living cells, at the single-virus particle or protein level. Here, we highlight the protocol for visualizing HIV-1 Gag assembly at the host T-cell plasma membrane using super-resolution light microscopy. Total internal reflection fluorescence microscopy (TIRF-M) coupled with single-molecule localization microscopy (SMLM) enables the detection and characterization of the assembly of viral proteins at the plasma membrane of infected host cells at the single protein level. Here, we describe the TIRF equipment, the T-cell culture for HIV-1, the sample preparation for single-molecule localization microscopies such as PALM and STORM, acquisition protocols, and Gag assembling cluster analysis.

A live cell imaging-based assay for tracking particle uptake by clathrin-mediated endocytosis.

A popular strategy for therapeutic delivery to cells and tissues is to encapsulate therapeutics inside particles that cells internalize via endocytosis. The efficacy of particle uptake by endocytosis is often studied in bulk using flow cytometry and Western blot analysis and confirmed using confocal microscopy. However, these techniques do not reveal the detailed dynamics of particle internalization and how the inherent heterogeneity of many types of particles may impact their endocytic uptake. Toward addressing these gaps, here we present a live-cell imaging-based method that utilizes total internal reflection fluorescence microscopy to track the uptake of a large ensemble of individual particles in parallel, as they interact with the cellular endocytic machinery. To analyze the resulting data, we employ an open-source tracking algorithm in combination with custom data filters. This analysis reveals the dynamic interactions between particles and endocytic structures, which determine the probability of particle uptake. In particular, our approach can be used to examine how variations in the physical properties of particles (size, targeting, rigidity), as well as heterogeneity within the particle population, impact endocytic uptake. These data impact the design of particles toward more selective and efficient delivery of therapeutics to cells.

Using Optical Tweezers Combined with Total Internal Reflection Microscopy to Study Interactions Between the ER and Golgi in Plant Cells.

Optical tweezers have been used to trap and micro-manipulate several biological specimens ranging from DNA, macromolecules, organelles, to single-celled organisms. Using a combination of the refraction and scattering of laser light from a focused laser beam, refractile objects are physically captured and can be moved within the surrounding media. The technique is routinely used to determine biophysical properties such as the forces exerted by motor proteins. Here, we describe how optical tweezers combined with total internal reflection fluorescence microscopy (TIRF) can be used to assess physical interactions between organelles, more specifically the ER and Golgi bodies in plant cells.

Reconstitution of Neuronal Motor Traffic on Septin-Associated Microtubules.

Reconstitution of intracellular transport in cell-free in vitro assays enables the understanding and dissection of the molecular mechanisms that underlie membrane traffic. Using total internal reflection fluorescence (TIRF) microscopy and microtubules, which are immobilized to a functionalized glass surface, the kinetic properties of single kinesin molecules can be imaged and analyzed in the presence or absence of microtubule-associated proteins. Here, we describe methods for the in vitro reconstitution of the motility of the neuronal kinesin motor KIF1A on microtubules associated with heteromeric septin (SEPT2/6/7) complexes. This method can be adapted for various neuronal septin complexes and kinesin motors, leading to new insights into the spatial regulation of neuronal membrane traffic by microtubule-associated septins.

Evaluation of functional transbilayer coupling in live cells by controlled lipid exchange and imaging fluorescence correlation spectroscopy.

Biophysical coupling between the inner and outer leaflets, known as inter-leaflet or transbilayer coupling, is a fundamental organizational principle in the plasma membranes of live mammalian cells. Lipid-based interactions between the two leaflets are proposed to be a primary mechanism underlying transbilayer coupling. However, there are only a few experimental evidence supporting the existence of such interactions in live cells. This is seemingly due to the lack of experimental strategies to perturb the lipid composition in one leaflet and quantitative techniques to evaluate the biophysical properties of the opposite leaflet. The existing strategies often dependent on immobilization and clustering a component in one of the leaflets and technically demanding biophysical tools to evaluate the effects on the opposing leaflet. In the recent years, the London group developed a simple but elegant method, namelymethyl-alpha-cyclodextrin catalyzed lipid exchange (LEX), to efficiently exchange outer leaflet lipids with an exogenous lipid of choice. Here, we adopted this method to perturb outer leaflet lipid composition. The corresponding changes in the inner leaflet is evaluated by comparing the diffusion of lipid probes localized in this leaflet in unperturbed and perturbed conditions. We employed highly multiplexed imaging fluorescence correlation spectroscopy (ImFCS), realized in a commercially available or home-built total internal reflection fluorescence microsocope equipped with a fast and sensitive camera, to determine diffusion coefficient of the lipid probes. Using the combination of LEX and ImFCS, we directly demonstrate lipid-based transbilayer coupling that does not require immobilization of membrane components in live mast cells in resting conditions. Overall, we present a relatively straightforward experimental strategy to evaluate transbilayer coupling quantitively in live cells.

Inner Front Cover Image

ON THE INNER FRONT COVER: Total internal reflection microscopy image of taxol‐stabilized microtubules coated in tau envelopes composed of human 2N4R tau (magenta). Tau envelopes exclude the binding of human MAP4 (green), resulting in distinct MAP‐covered domains along single microtubules in vitro.Credit: Richard J. McKenney, University of California, Davis.image

Zwitterionic Polysulfobetaine Inhibits Cancer Cell Migration Owing to Actin Cytoskeleton Dynamics.

Cell migration is an essential dynamic process for most living cells, mainly driven by the reorganization of actin cytoskeleton. To control actin dynamics, a molecular architecture that can serve as a nucleator has been designed by polymerizing sulfobetaine methacrylate. The synthesized zwitterionic polymer, poly(sulfobetaine methacrylate) (PZI), effectively nucleates the polymerization process of G-actin and substantially accelerates the rate of polymerization. Isothermal titration calorimetry (ITC) and bioinformatics analysis indicated binding between PZI and monomeric G-actin. Thus, in vitro actin dynamics was studied by dynamic light scattering (DLS), pyrene-actin polymerization assay, and total internal reflection fluorescence microscopy (TIRFM). Furthermore, a 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene (BODIPY) fluorophore-containing monomeric unit was incorporated into the sulfobetaine zwitterionic architecture to visualize the effect of polymer in the cellular environment. The BODIPY-containing zwitterionic sulfobetaine polymer (PZI-F) successfully penetrated the cell and remained in the lysosome with minimal cytotoxicity. Confocal microscopy revealed the influence of this polymer on the cellular actin cytoskeleton dynamics. The PZI-F polymer was successfully able to inhibit the collective migration of the human cervical cancer cell line (HeLa cell) and breast cancer cell line (MDA-MB-231 cell), as confirmed by a wound healing assay. Therefore, polyzwitterionic sulfobetaine could be explored as an inhibitor of cancer cell migration.

Drebrin Protects Assembled Actin from INF2-FFC-mediated Severing and Stabilizes Cell Protrusions

Highly specialized cells, such as neurons and podocytes, have arborized morphologies that serve their specific functions. Actin cytoskeleton and its associated proteins are responsible for the distinctive shapes of cells. The mechanism of their cytoskeleton regulation – contributing to cell shape maintenance – is yet to be fully clarified. Inverted formin 2 (INF2), one of the modulators of the cytoskeleton, is an atypical formin that can both polymerize and depolymerize actin filaments depending on its molar ratio to actin. Prior work has established that INF2 binds to the sides of actin filaments and severs them. Drebrin is another actin-binding protein that also binds filaments laterally and stabilizes them, but the interplay between drebrin and INF2 on actin filament stabilization is not well understood. Here, we have used biochemical assays, electron microscopy, and total internal reflection fluorescence microscopy imaging to show that drebrin protects actin filaments from severing by INF2 without inhibiting its polymerization activity. Notably, truncated drebrin – DrbA1-300 – is sufficient for this protection, though not as effective as the full-length protein. INF2 and drebrin are abundantly expressed in highly specialized cells and are crucial for the temporal regulation of their actin cytoskeleton, consistent with their involvement in peripheral neuropathy.

Real-time imaging of individual electropores proves their longevity in cells

With over 50 years of electroporation research, the nature of cell membrane permeabilization remains elusive. The lifetime of electropores in molecular models is limited to nano- or microseconds, whereas the permeabilization of electroporated cells can last minutes. This study aimed at resolving a longstanding debate on whether the prolonged permeabilization is due to the formation of long-lived pores in cells. We developed a method for dynamic monitoring and conductance measurements of individual electropores. This was accomplished by time-lapse total internal reflection fluorescence (TIRF) imaging in HEK cells loaded with CAL-520 dye and placed on an indium tin oxide (ITO) surface. Applying a 1-ms, 0 to −400 mV pulse between the patch pipette and ITO evoked focal Ca2+ transients that identified individual electropores. Some transients disappeared in milliseconds but others persisted for over a minute. Persistent transients (“Ca2+ plumes”) faded over time to a stable or a randomly fluctuating level that could include periods of full quiescence. Single pore conductance, measured by 0 to −50 mV, 50 ms steps at 30 and 60 s after the electroporation, ranged from 80 to 200 pS. These experiments proved electropore longevity in cells, in stark contrast to molecular simulations and many findings in lipid bilayers.

GLP-1 metabolite GLP-1(9–36) is a systemic inhibitor of mouse and human pancreatic islet glucagon secretion

Aims/hypothesisDiabetes mellitus is associated with impaired insulin secretion, often aggravated by oversecretion of glucagon. Therapeutic interventions should ideally correct both defects. Glucagon-like peptide 1 (GLP-1) has this capability but exactly how it exerts its glucagonostatic effect remains obscure. Following its release GLP-1 is rapidly degraded from GLP-1(7–36) to GLP-1(9–36). We hypothesised that the metabolite GLP-1(9–36) (previously believed to be biologically inactive) exerts a direct inhibitory effect on glucagon secretion and that this mechanism becomes impaired in diabetes.MethodsWe used a combination of glucagon secretion measurements in mouse and human islets (including islets from donors with type 2 diabetes), total internal reflection fluorescence microscopy imaging of secretory granule dynamics, recordings of cytoplasmic Ca2+ and measurements of protein kinase A activity, immunocytochemistry, in vivo physiology and GTP-binding protein dissociation studies to explore how GLP-1 exerts its inhibitory effect on glucagon secretion and the role of the metabolite GLP-1(9–36).ResultsGLP-1(7–36) inhibited glucagon secretion in isolated islets with an IC50 of 2.5 pmol/l. The effect was particularly strong at low glucose concentrations. The degradation product GLP-1(9–36) shared this capacity. GLP-1(9–36) retained its glucagonostatic effects after genetic/pharmacological inactivation of the GLP-1 receptor. GLP-1(9–36) also potently inhibited glucagon secretion evoked by β-adrenergic stimulation, amino acids and membrane depolarisation. In islet alpha cells, GLP-1(9–36) led to inhibition of Ca2+ entry via voltage-gated Ca2+ channels sensitive to ω-agatoxin, with consequential pertussis-toxin-sensitive depletion of the docked pool of secretory granules, effects that were prevented by the glucagon receptor antagonists REMD2.59 and L-168049. The capacity of GLP-1(9–36) to inhibit glucagon secretion and reduce the number of docked granules was lost in alpha cells from human donors with type 2 diabetes. In vivo, high exogenous concentrations of GLP-1(9–36) (>100 pmol/l) resulted in a small (30%) lowering of circulating glucagon during insulin-induced hypoglycaemia. This effect was abolished by REMD2.59, which promptly increased circulating glucagon by >225% (adjusted for the change in plasma glucose) without affecting pancreatic glucagon content.Conclusions/interpretationWe conclude that the GLP-1 metabolite GLP-1(9–36) is a systemic inhibitor of glucagon secretion. We propose that the increase in circulating glucagon observed following genetic/pharmacological inactivation of glucagon signalling in mice and in people with type 2 diabetes reflects the removal of GLP-1(9–36)’s glucagonostatic action.Graphical