Search Results (25)

Total internal reflection fluorescence (TIRF) microscopy excites fluorophores within a few hundred nanometers of the sample–substrate interface, enabling high-contrast imaging near the cell membrane. When cultured cells differentiate, the membrane in contact with the coverslip generally acquires basal characteristics, while the opposite membrane develops apical features. Consequently, conventional TIRF microscopy is limited to imaging the basal surface. We developed an immersed-prism TIRF (IP-TIRF) microscope, in which a prism immersed in the culture medium generates TIR at the cell/medium–prism interface, illuminating the apical membrane and reducing cytosolic background. In proof-of-principle experiments, we imaged fluorescent beads and 3xmNeonGreen-tagged intraflagellar transport (IFT) particles in cilia, and compared the performance with confocal microscopy. In cellular regions where both methods can be applied (such as the IFT base pool), on average, IP-TIRF achieved approximately 1.8 times the contrast-to-noise ratio (CNR~31) compared to confocal microscopy. Furthermore, IFT-particle motion was detected in IP-TIRF image sequences and Kymographs of cilia, with adequate spatial resolution. Kymograph analysis revealed an average anterograde IFT velocity of 0.156 ± 0.071 µm/s and an average retrograde velocity of 0.020 ± 0.007 µm/s, approximately one-quarter and one-twentieth, respectively, of the values reported for mammalian primary cilia, which we attribute to acquisition at room temperature rather than physiological conditions. Therefore, these velocity measurements should be regarded as proof-of-principle demonstrations obtained at room temperature, not as validated physiological transport rates. Our IP-TIRF method provides a high-resolution, cost-effective, and broadly accessible approach for imaging the apical membrane in live cells. Full article

Total internal reflection fluorescence–structured illumination microscopy (TIRF-SIM) can enhance the lateral resolution of fluorescence microscopy to twice the diffraction limit, enabling subtler observations of activity in subcellular life. However, the lack of an axial resolution makes it difficult to resolve three-dimensional (3D) subcellular structures. In this paper, we present an alternative TIRF-SIM axial resolution enhancement method by exploiting quantitative information regarding the distance between fluorophores and the surface within the evanescent field. Combining the lateral super-resolution information of TIRF-SIM with reconstructed axial information, a 3D super-resolution image with a 25 nm axial resolution is achieved without attaching special optical components or high-power lasers. The reconstruction results of cell samples demonstrate that the axial resolution enhancement method for TIRF-SIM can effectively resolve the axial depth of densely structured regions. Full article

Single-chain polymeric nanoparticles (SCPNs) have been extensively explored as a synthetic alternative to enzymes for catalytic applications. However, the inherent structural heterogeneity of SCPNs, arising from the dispersity of the polymer backbone and stochastic incorporation of different monomers as well as catalytic moieties, is expected to lead to variations in catalytic activity between individual particles. To understand the effect of structural heterogeneities on the catalytic performance of SCPNs, techniques are required that permit researchers to directly monitor SCPN activity at the single-polymer level. In this study, we introduce the use of single-molecule fluorescence microscopy to study the kinetics of Cu(I)-containing SCPNs towards depropargylation reactions. We developed Cu(I)-containing SCPNs that exhibit fast kinetics towards depropargylation and Cu-catalyzed azide-alkyne click reactions, making them suitable for single-particle kinetic studies. SCPNs were then immobilized on the surface of glass coverslips and the catalytic reactions were monitored at a single-particle level using total internal reflection fluorescence (TIRF) microscopy. Our studies revealed the interparticle turnover dispersity for Cu(I)-catalyzed depropargylations. In the future, our approach can be extended to different polymer designs which can give insights into the intrinsic heterogeneity of SCPN catalysis and can further aid in the rational development of SCPN-based catalysts. Full article

Type-2 ryanodine receptor (RyR2) is the major Ca²⁺ release channel of the cardiac sarcoplasmic reticulum (SR) that controls the rhythm and strength of the heartbeat, but its malfunction may generate severe arrhythmia leading to sudden cardiac death or heart failure. S4938F-RyR2 mutation in the carboxyl-terminal was expressed in human induced pluripotent stem cells derived cardiomyocytes (hiPSC-CMs) using CRISPR/Cas9 gene-editing technique. Ca²⁺ signaling and electrophysiological properties of beating cardiomyocytes carrying the mutation were studied using total internal reflection fluorescence microscopy (TIRF) and patch clamp technique. In mutant cells, L-type Ca²⁺ currents (I_Ca), measured either by depolarizations to zero mV or repolarizations from +100 mV to –50 mV, and their activated Ca²⁺ transients were significantly smaller, despite their larger caffeine-triggered Ca²⁺ release signals compared to wild type (WT) cells, suggesting I_Ca-induced Ca²⁺ release (CICR) was compromised. The larger SR Ca²⁺ content of S4938F-RyR2 cells may underlie the higher frequency of spontaneously occurring Ca²⁺ sparks and Ca²⁺ transients and their arrhythmogenic phenotype. Full article

Single-molecule imaging technologies, especially those based on fluorescence, have been developed to probe both the equilibrium and dynamic properties of biomolecules at the single-molecular and quantitative levels. In this review, we provide an overview of the state-of-the-art advancements in single-molecule fluorescence imaging techniques. We systematically explore the advanced implementations of in vitro single-molecule imaging techniques using total internal reflection fluorescence (TIRF) microscopy, which is widely accessible. This includes discussions on sample preparation, passivation techniques, data collection and analysis, and biological applications. Furthermore, we delve into the compatibility of microfluidic technology for single-molecule fluorescence imaging, highlighting its potential benefits and challenges. Finally, we summarize the current challenges and prospects of fluorescence-based single-molecule imaging techniques, paving the way for further advancements in this rapidly evolving field. Full article

Actin polymerization drives cell movement and provides cells with structural integrity. Intracellular environments contain high concentrations of solutes, including organic compounds, macromolecules, and proteins. Macromolecular crowding has been shown to affect actin filament stability and bulk polymerization kinetics. However, the molecular mechanisms behind how crowding influences individual actin filament assembly are not well understood. In this study, we investigated how crowding modulates filament assembly kinetics using total internal reflection fluorescence (TIRF) microscopy imaging and pyrene fluorescence assays. The elongation rates of individual actin filaments analyzed from TIRF imaging depended on the type of crowding agent (polyethylene glycol, bovine serum albumin, and sucrose) as well as their concentrations. Further, we utilized all-atom molecular dynamics (MD) simulations to evaluate the effects of crowding molecules on the diffusion of actin monomers during filament assembly. Taken together, our data suggest that solution crowding can regulate actin assembly kinetics at the molecular level. Full article

T7 phages are E. coli-infecting viruses that find and invade their target with high specificity and efficiency. The exact molecular mechanisms of the T7 infection cycle are yet unclear. As the infection involves mechanical events, single-particle methods are to be employed to alleviate the problems of ensemble averaging. Here we used TIRF microscopy to uncover the spatial dynamics of the target recognition and binding by individual T7 phage particles. In the initial phase, T7 virions bound reversibly to the bacterial membrane via two-dimensional diffusive exploration. Stable bacteriophage anchoring was achieved by tail-fiber complex to receptor binding which could be observed in detail by atomic force microscopy (AFM) under aqueous buffer conditions. The six anchored fibers of a given T7 phage-displayed isotropic spatial orientation. The viral infection led to the onset of an irreversible structural program in the host which occurred in three distinct steps. First, bacterial cell surface roughness, as monitored by AFM, increased progressively. Second, membrane blebs formed on the minute time scale (average ~5 min) as observed by phase-contrast microscopy. Finally, the host cell was lysed in a violent and explosive process that was followed by the quick release and dispersion of the phage progeny. DNA ejection from T7 could be evoked in vitro by photothermal excitation, which revealed that genome release is mechanically controlled to prevent premature delivery of host-lysis genes. The single-particle approach employed here thus provided an unprecedented insight into the details of the complete viral cycle. Full article

Single-molecule total internal reflection fluorescence (TIRF) microscopy allows for the real-time visualization of macromolecular dynamics and complex assembly. Prism-based TIRF microscopes (prismTIRF) are relatively simple to operate and can be easily modulated to fit the needs of a wide variety of experimental applications. While building a prismTIRF microscope without expert assistance can pose a significant challenge, the components needed to build a prismTIRF microscope are relatively affordable and, with some guidance, the assembly can be completed by a determined novice. Here, we provide an easy-to-follow guide for the design, assembly, and operation of a three-color prismTIRF microscope which can be utilized for the study of macromolecular complexes, including the multi-component protein–DNA complexes responsible for DNA repair, replication, and transcription. Our hope is that this article can assist laboratories that aspire to implement single-molecule TIRF techniques, and consequently expand the application of this technology. Full article

This review highlights the significant observations of human immunodeficiency virus (HIV) assembly, release and maturation made possible with advanced light microscopy techniques. The advances in technology which now enables these light microscopy measurements are discussed with special emphasis on live imaging approaches including Total Internal Reflection Fluorescence (TIRF), high-resolution light microscopy techniques including PALM and STORM and single molecule measurements, including Fluorescence Resonance Energy Transfer (FRET). The review concludes with a discussion on what new insights and understanding can be expected from these measurements. Full article

Due to the global rise of type 2 diabetes mellitus (T2DM) in combination with insulin resistance, novel compounds to efficiently treat this pandemic disease are needed. Screening for compounds that induce the translocation of glucose transporter 4 (GLUT4) from the intracellular compartments to the plasma membrane in insulin-sensitive tissues is an innovative strategy. Here, we compared the applicability of three fluorescence microscopy-based assays optimized for the quantitation of GLUT4 translocation in simple cell systems. An objective-type scanning total internal reflection fluorescence (TIRF) microscopy approach was shown to have high sensitivity but only moderate throughput. Therefore, we implemented a prism-type TIR reader for the simultaneous analysis of large cell populations grown in adapted microtiter plates. This approach was found to be high throughput and have sufficient sensitivity for the characterization of insulin mimetic compounds in live cells. Finally, we applied confocal microscopy to giant plasma membrane vesicles (GPMVs) formed from GLUT4-expressing cells. While this assay has only limited throughput, it offers the advantage of being less sensitive to insulin mimetic compounds with high autofluorescence. In summary, the combined implementation of different fluorescence microscopy-based approaches enables the quantitation of GLUT4 translocation with high throughput and high content. Full article

Here, we present a simple and robust experimental setup for the super-resolution live cell microscopy of membrane-proximal fluorophores, which is comparably easy to perform and to implement. The method is based on Structured Illumination Microscopy (SIM) with a switchable spatial light modulator (SLM) and exchangeable objective lenses for epi-illumination and total internal reflection fluorescence (TIRF) microscopy. While, in the case of SIM (upon epi-illumination), cell layers of about 1–2 µm in close proximity to the plasma membrane can be selected by software, layers in the 100 nm range are assessed experimentally by TIRF-SIM. To show the applicability of this approach, both methods are used to measure the translocation of the glucose transporter 4 (GLUT4) from intracellular vesicles to the plasma membrane upon stimulation by insulin or insulin-mimetic compounds, with a lateral resolution of around 100 nm and an axial resolution of around 200 nm. While SIM is an appropriate method to visualize the intracellular localization of GLUT4 fused with a green fluorescent protein, TIRF-SIM permits the quantitative evaluation of its fluorescence in the plasma membrane. These imaging methods are discussed in the context of fluorescence lifetime kinetics, providing additional data for the molecular microenvironment. Full article

The Ca²⁺-permeable Transient Receptor Potential channel vanilloid subfamily member 4 (TRPV4) is involved in a broad range of physiological processes, including the regulation of systemic osmotic pressure, bone resorption, vascular tone, and bladder function. Mutations in the TRPV4 gene are the cause of a spectrum of inherited diseases (or TRPV4-pathies), which include skeletal dysplasias, arthropathies, and neuropathies. There is little understanding of the pathophysiological mechanisms underlying these variable disease phenotypes, but it has been hypothesized that disease-causing mutations affect interaction with regulatory proteins. Here, we performed a mammalian protein–protein interaction trap (MAPPIT) screen to identify proteins that interact with the cytosolic N terminus of human TRPV4, a region containing the majority of disease-causing mutations. We discovered the zinc-finger domain-containing protein ZC4H2 as a TRPV4-interacting protein. In heterologous expression experiments, we found that ZC4H2 increases both the basal activity of human TRPV4 as well as Ca²⁺ responses evoked by ligands or hypotonic cell swelling. Using total internal reflection fluorescence (TIRF) microscopy, we further showed that ZC4H2 accelerates TRPV4 turnover at the plasma membrane. Overall, these data demonstrate that ZC4H2 is a positive modulator of TRPV4, and suggest a link between TRPV4 and ZC4H2-associated rare disorders, which have several neuromuscular symptoms in common with TRPV4-pathies. Full article

An urgent demand exists for the development of novel delivery systems that efficiently transport antibacterial agents across cellular membranes for the eradication of intracellular pathogens. In this study, the clinically relevant poorly water-soluble antibiotic, rifampicin, was confined within mesoporous silica nanoparticles (MSN) to investigate their ability to serve as an efficacious nanocarrier system against small colony variants of Staphylococcus aureus (SCV S. aureus) hosted within Caco-2 cells. The surface chemistry and particle size of MSN were varied through modifications during synthesis, where 40 nm particles with high silanol group densities promoted enhanced cellular uptake. Extensive biophysical analysis was performed, using quartz crystal microbalance with dissipation (QCM-D) and total internal reflection fluorescence (TIRF) microscopy, to elucidate the mechanism of MSN adsorption onto semi-native supported lipid bilayers (snSLB) and, thus, uncover potential cellular uptake mechanisms of MSN into Caco-2 cells. Such studies revealed that MSN with reduced silanol group densities were prone to greater particle aggregation on snSLB, which was expected to restrict endocytosis. MSN adsorption and uptake into Caco-2 cells correlated well with antibacterial efficacy against SCV S. aureus, with 40 nm hydrophilic particles triggering a ~2.5-log greater reduction in colony forming units, compared to the pure rifampicin. Thus, this study provides evidence for the potential to design silica nanocarrier systems with controlled surface chemistries that can be used to re-sensitise intracellular bacteria to antibiotics by delivering them to the site of infection. Full article

Cancer heterogeneity increasingly requires ultrasensitive techniques that allow early diagnosis for personalized treatment. In addition, they should preferably be non-invasive tools that do not damage surrounding tissues or contribute to body toxicity. In this context, liquid biopsy of biological samples such as urine, blood, or saliva represents an ideal approximation of what is happening in real time in the affected tissues. Plasmonic nanoparticles are emerging as an alternative or complement to current diagnostic techniques, being able to detect and quantify novel biomarkers such as specific peptides and proteins, microRNA, circulating tumor DNA and cells, and exosomes. Here, we review the latest ideas focusing on the use of plasmonic nanoparticles in coded and label-free surface-enhanced Raman scattering (SERS) spectroscopy. Moreover, surface plasmon resonance (SPR) spectroscopy, colorimetric assays, dynamic light scattering (DLS) spectroscopy, mass spectrometry or total internal reflection fluorescence (TIRF) microscopy among others are briefly examined in order to highlight the potential and versatility of plasmonics. Full article

Peptide and protein micropatterns are powerful tools for the investigation of various cellular processes, including protein–protein interactions (PPIs). Within recent years, various approaches for the production of functional surfaces have been developed. Most of these systems use glass as a substrate, which has several drawbacks, including high fragility and costs, especially if implemented for fluorescence microscopy. In addition, conventional fabrication technologies such as microcontact printing (µCP) are frequently used for the transfer of biomolecules to the glass surface. In this case, it is challenging to adjust the biomolecule density. Here, we show that cyclic olefin polymer (COP) foils, with their encouraging properties, including the ease of manufacturing, chemical resistance, biocompatibility, low water absorption, and optical clarity, are a promising alternative to glass substrates for the fabrication of micropatterns. Using a photolithography-based approach, we generated streptavidin/biotinylated antibody patterns on COPs with the possibility of adjusting the pattern contrast by varying plasma activation parameters. Our experimental setup was finally successfully implemented for the analysis of PPIs in the membranes of live cells via total internal reflection fluorescence (TIRF) microscopy. Full article