Total Internal Reflection Fluorescence Research Articles

Extracellular-Vesicle Catch-and-Release Isolation System Using a Net-Charge Invertible Curvature-Sensing Peptide.

Extracellular vesicles (EVs) carry various informative components, including signaling proteins, transcriptional regulators, lipids, and nucleic acids. These components are utilized for cell-cell communication between donor and recipient cells. EVs have shown great promise as pharmaceutical-targeting vesicles and have attracted the attention of researchers in the fields of biological and medical science because of their importance as diagnostic and prognostic markers. However, the isolation and purification of EVs from cell-cultured media remain challenging. Ultracentrifugation is the most widely used method, but it requires specialized and expensive equipment. In the present study, we proposed a novel methodology to isolate EVs using a simple and convenient method, i.e., an EV catch-and-release isolation system (EV-CaRiS) using a net-charge invertible curvature-sensing peptide (NIC). Curvature-sensing peptides recognize vesicles by binding to lipid-packing defects on highly curved membranes regardless of the expression levels of biomarkers. NIC was newly designed to reversibly capture and release EVs in a pH-dependent manner. NIC allowed us to achieve reproducible EV isolation from three human cell lines on resin using a batch method and single-particle imaging of EVs containing the ubiquitous exosome markers CD63 and CD81 by total internal reflection fluorescence microscopy (TIRFM). EV-CaRiS was demonstrated as a simple and convenient methodology for EV isolation, and NIC is promising for applications in the single-particle analysis of EVs.

Mosaic quadrivalent influenza vaccine single nanoparticle characterization

Recent work by our laboratory and others indicates that co-display of multiple antigens on protein-based nanoparticles may be key to induce cross-reactive antibodies that provide broad protection against disease. To reach the ultimate goal of a universal vaccine for seasonal influenza, a mosaic influenza nanoparticle vaccine (FluMos-v1) was developed for clinical trial (NCT04896086). FluMos-v1 is unique in that it is designed to co-display four recently circulating haemagglutinin (HA) strains; however, current vaccine analysis techniques are limited to nanoparticle population analysis, thus, are unable to determine the valency of an individual nanoparticle. For the first time, we demonstrate by total internal reflection fluorescence microscopy and supportive physical–chemical methods that the co-display of four antigens is indeed achieved in single nanoparticles. Additionally, we have determined percentages of multivalent (mosaic) nanoparticles with four, three, or two HA proteins. The integrated imaging and physicochemical methods we have developed for single nanoparticle multivalency will serve to further understand immunogenicity data from our current FluMos-v1 clinical trial.

Probing Activation and Conformational Dynamics of the Vesicle-Reconstituted β2 Adrenergic Receptor at the Single-Molecule Level.

G-protein-coupled receptors (GPCRs) are structurally flexible membrane proteins that mediate a host of physiological responses to extracellular ligands like hormones and neurotransmitters. Fine features of their dynamic structural behavior are hypothesized to encode the functional plasticity seen in GPCR activity, where ligands with different efficacies can direct the same receptor toward different signaling phenotypes. Although the number of GPCR crystal structures is increasing, the receptors are characterized by complex and poorly understood conformational landscapes. Therefore, we employed a fluorescence microscopy assay to monitor conformational dynamics of single β2 adrenergic receptors (β2ARs). To increase the biological relevance of our findings, we decided not to reconstitute the receptor in detergent micelles but rather lipid membranes as proteoliposomes. The conformational dynamics were monitored by changes in the intensity of an environmentally sensitive boron-dipyrromethene (BODIPY 493/503) fluorophore conjugated to an endogenous cysteine (located at the cytoplasmic end of the sixth transmembrane helix of the receptor). Using total internal reflection fluorescence microscopy (TIRFM) and a single small unilamellar liposome assay that we previously developed, we followed the real-time dynamic properties of hundreds of single β2ARs reconstituted in a native-like environment─lipid membranes. Our results showed that β2AR-BODIPY fluctuates between several states of different intensity on a time scale of seconds, compared to BODIPY-lipid conjugates that show almost entirely stable fluorescence emission in the absence and presence of the full agonist BI-167107. Agonist stimulation changes the β2AR dynamics, increasing the population of states with higher intensities and prolonging their durations, consistent with bulk experiments. The transition density plot demonstrates that β2AR-BODIPY, in the absence of the full agonist, interconverts between states of low and moderate intensity, while the full agonist renders transitions between moderate and high-intensity states more probable. This redistribution is consistent with a mechanism of conformational selection and is a promising first step toward characterizing the conformational dynamics of GPCRs embedded in a lipid bilayer.

Analysis of Poly(ethylene terephthalate) degradation kinetics of evolved IsPETase variants using a surface crowding model

Poly(ethylene terephthalate) (PET) is a major plastic polymer utilized in the single-use and textile industries. The discovery of PET-degrading enzymes (PETases) has led to an increased interest in the biological recycling of PET in addition to mechanical recycling. IsPETase from Ideonella sakaiensis is a candidate catalyst, but little is understood about its structure-function relationships with regards to PET degradation. To understand the effects of mutations on IsPETase productivity, we develop a directed evolution assay to identify mutations beneficial to PET film degradation at 30ºC. IsPETase also displays enzyme concentration-dependent inhibition effects, and surface crowding has been proposed as a causal phenomenon. Based on total internal reflectance fluorescence microscopy and adsorption experiments, IsPETase is likely experiencing crowded conditions on PET films. Molecular dynamics (MD) simulations of IsPETase variants reveal a decrease in active site flexibility in free enzymes and reduced probability of productive active site formation in substrate-bound enzymes under crowding. Hence, we develop a surface crowding model to analyze the biochemical effects of three hit mutations (T116P, S238N, S290P) that enhanced ambient temperature activity and/or thermostability. We find that T116P decreases susceptibility to crowding, resulting in higher PET degradation product accumulation despite no change in intrinsic catalytic rate. In conclusion, we show that a macromolecular crowding-based biochemical model can be used to analyze the effects of mutations on properties of PETases and that crowding behavior is a major property to be targeted for enzyme engineering for improved PET degradation.

Pre-fusion motion state determines the heterogeneity of membrane fusion dynamics for large dense-core vesicles.

In neuroendocrine cells, large dense-core vesicles (LDCVs) undergo highly regulated pre-fusion processes before releasing hormones via membrane fusion. Significant heterogeneity has been found for LDCV population based on the dynamics of membrane fusion. However, how the pre-fusion status impacts the heterogeneity of LDCVs still remains unclear. Hence, we explored pre-fusion determinants of heterogeneous membrane fusion procedure of LDCV subpopulations. We assessed the pre-fusion motion of two LDCV subpopulations with distinct membrane fusion dynamics individually, using total internal reflection fluorescence microscopy. These two subpopulations were isolated by blocking Rho GTPase-dependent actin reorganization using Clostridium difficile toxin B (ToxB), which selectively targets the fast fusion vesicle pool. We found that the fast fusion subpopulation was in an active motion mode prior to release, termed "active" LDCV pool, while vesicles from the slow fusion subpopulation were also moving but in a significantly more confined status, forming an "inert" pool. The depletion of the active pool by ToxB also eliminated fast fusion vesicles and was not rescued by pre-treatment with phorbol ester. A mild actin reorganization blocker, latrunculin A, that partially disrupted the active pool, only slightly attenuated the fast fusion subpopulation. The pre-fusion motion state of LDCVs also exhibits heterogeneity and dictates the heterogeneous fusion pore dynamics. Rearrangement of F-actin network mediates vesicle pre-fusion motion and subsequently determines the membrane fusion kinetics.

Unveiling PET Hydrolase Surface Dynamics through Fluorescence Microscopy.

PET hydrolases are an emerging class of enzymes that are being heavily researched for their use in bioprocessing polyethylene terephthalate (PET). While work has been done in studying the binding of PET oligomers to the active site of these enzymes, the dynamics of PET hydrolases binding to a bulk PET surface is an unexplored area. Here, methods were developed for total internal reflection fluorescence (TIRF) microscopy and fluorescence recovery after photobleaching (FRAP) microscopy to study the adsorption and desorption dynamics of these proteins onto a PET surface. TIRF microscopy was employed to measure both on and off rates of two of the most commonly studied PET hydrolases, PHL7 and LCC, on a PET surface. It was found that these proteins have a much slower off rates on the order of 10-3 s-1 , comparable to non-productive binding in enzymes such as cellulose. In combination with FRAP microscopy, a dynamic model is proposed in which adsorption and desorption dominates over lateral diffusion over the surface. The results of this study could have implications for the future engineering of PET hydrolases, either to target them to a PET surface or to modulate interaction with their substrate.

Retention of the RNA ends provides the molecular memory for maintaining the activation of the Csm complex.

The type III CRISPR-Cas effector complex Csm functions as a molecular Swiss army knife that provides multilevel defense against foreign nucleic acids. The coordinated action of three catalytic activities of the Csm complex enables simultaneous degradation of the invader's RNA transcripts, destruction of the template DNA and synthesis of signaling molecules (cyclic oligoadenylates cAn) that activate auxiliary proteins to reinforce CRISPR-Cas defense. Here, we employed single-molecule techniques to connect the kinetics of RNA binding, dissociation, and DNA hydrolysis by the Csm complex from Streptococcus thermophilus. Although single-stranded RNA is cleaved rapidly (within seconds), dual-color FCS experiments and single-molecule TIRF microscopy revealed that Csm remains bound to terminal RNA cleavage products with a half-life of over 1 hour while releasing the internal RNA fragments quickly. Using a continuous fluorescent DNA degradation assay, we observed that RNA-regulated single-stranded DNase activity decreases on a similar timescale. These findings suggest that after fast target RNA cleavage the terminal RNA cleavage products stay bound within the Csm complex, keeping the Cas10 subunit activated for DNA destruction. Additionally, we demonstrate that during Cas10 activation, the complex remains capable of RNA turnover, i.e. of ongoing degradation of target RNA.

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.

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

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.