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A schematic illustration of the flow-cell manufacturing process. (a) Double sided tape is placed on a pre-cleaned glass microscope slide containing 1 inlet and 1 outlet hole for sample injection. A pre-cleaned glass microscope coverslip is added directly on top of the double-sided tape to form a chamber. (b) After coating the interior of the chamber with poly-l-lysine and the injection of pre-formed Aβ 555 complexes, the fluidic chamber is placed coverslip down onto an oil immersion objective lens housed in an inverted microscope to enable monitoring of surface immobilized oligomers.
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A major hallmark of Alzheimer’s disease is the misfolding and aggregation of the amyloid- β peptide (A β). While early research pointed towards large fibrillar- and plaque-like aggregates as being the most toxic species, recent evidence now implicates small soluble A β oligomers as being orders of magnitude more harmful. Techniques capable of chara...
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Context 1
... stepwise photobleaching trajectories were obtained from Aβ 555 oligomers immobilized onto the surface of a pre-cleaned glass coverslip (22 × 50 mm, No. 1.5 thickness, Scientific Laboratory Supplies Ltd, part number MIC3246), which formed part of a ~1 cm wide microscope flow-cell. A schematic illustration of the flow-cell is shown in Fig. 4. The text that follows provides a step-by-step guide to fabricating flow-cells and performing oligomer ...
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... begin the process of preparing a flow-cell, first drill two holes (ϕ = 1 mm) in 25 × 75 × 1 mm glass microscope slides (Polysciences Inc., part number 22245) for the purposes of sample injection into the flow-cell via pipette tip as schematically shown in Fig. 4a. It is worth noting that flow-cells were typically prepared in batches of five. Small diamond core drill bits (Eternal Tools, UK, part number SDCD1100) were used to drill 1 inlet and 1 outlet hole per slide. Prior to flow-cell fabrication, each pre-drilled microscope slide and glass coverslip must then be thoroughly cleaned as per ...
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... fabricate flow-cells by applying double-sided tape to the precleaned microscope slide as demonstrated in Fig. 4a in order to construct the walls of the chamber. Care should be taken to ensure a channel thickness of ~1 cm. Apply a pre-cleaned glass coverslip on top to form the ceiling of the chamber and gently push it against the tape using a pipette tip to complete fabrication. Remove residual tape carefully with a razor blade and seal the edges ...
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Super-resolution imaging based on single molecule localization of cellular structures on nanometer scale requires to record a series of wide-field or TIRF images resulting in a considerable recording time (typically of minutes). Therefore, sample drift becomes a critical problem and will lower the imaging precision. Herein we utilized morphological...
Citations
... As highlighted in Figure 2D, CV allows for the monitoring of the evolution of oligomer populations with a temporal resolution not feasible with antibodies, and concurrently with fibril formation. CV should also simplify the detection and quantification of oligomer populations in high-resolution optical microscopy, such as total internal reflection microscopy [52] or superresolution microscopy [53]. This should help address the question of whether the large populations of amyloid oligomers readily observed in vitro are on-pathway precursors or off-pathway competitors of fibril formation [54]. ...
Deposition of extracellular Amyloid Beta (Aβ) and intracellular tau fibrils in post-mortem brains remains the only way to conclusively confirm cases of Alzheimer’s Disease (AD). Substantial evidence, though, implicates small globular oligomers instead of fibrils as relevant biomarkers of, and critical contributors to, the clinical symptoms of AD. Efforts to verify and utilize amyloid oligomers as AD biomarkers in vivo have been limited by the near-exclusive dependence on conformation-selective antibodies for oligomer detection. While antibodies have yielded critical evidence for the role of both Aβ and tau oligomers in AD, they are not suitable for imaging amyloid oligomers in vivo. Therefore, it would be desirable to identify a set of oligomer-selective small molecules for subsequent development into Positron Emission Tomography (PET) probes. Using a kinetics-based screening assay, we confirm that the triarylmethane dye Crystal Violet (CV) is oligomer-selective for Aβ42 oligomers (AβOs) grown under near-physiological solution conditions in vitro. In postmortem brains of an AD mouse model and human AD patients, we demonstrate that A11 antibody-positive oligomers but not Thioflavin S (ThioS)-positive fibrils colocalize with CV staining, confirming in vitro results. Therefore, our kinetic screen represents a robust approach for identifying new classes of small molecules as candidates for oligomer-selective dyes (OSDs). Such OSDs, in turn, provide promising starting points for the development of PET probes for pre-mortem imaging of oligomer deposits in humans.
... i) the second convex lens in the expansion telescope was mounted on a lateral translation stage with a high-precision micrometer; this shift (up to 2.3 mm) generated an equivalent lateral displacement of the beam at the 6 mm diameter objective back aperture, thereby tilting the beam away from the optic axis at the sample. The beam delivery angle was calibrated for lens micrometer position following [78] and set to 50° ± 3° from normal incidence in oil, corresponding to 60° ± 5° in water by Snell's Law, with minimal coupling into the evanescent field at the coverslip. ii) a field stop was placed in the conjugate plane upstream of the first telescope lens. ...
Epigenetic regulation maintains gene expression patterns over many rounds of cell division in higher organisms. However, visualization of factors regulating epigenetic switches in vivo is limited by the challenge of imaging cells deep in living tissue, with molecular sensitivity and rapid sampling. We report an easy-to-implement method called Variable-angle Slimfield microscopy (SlimVar), which by simple modification of an inverted optical microscope, enables single-molecule tracking of fluorescent reporters in Arabidopsis thaliana. Using SlimVar, we imaged stepwise photobleaching of chromatin-protein assemblies in individual nuclei, 30 microns deep in root tips through multiple cell layers. We find that two homologous proteins key to the epigenetic switch at FLOWERING LOCUS C (FLC) - cold-induced VERNALIZATION INSENSITIVE3 (VIN3) and constitutively expressed VERNALIZATION 5 (VRN5) - exhibit dynamic nuclear assemblies during FLC silencing. Upon cold exposure, these assemblies increase in stoichiometry by up to 100% to a median of ~20 molecules. Larger VRN5 assemblies preferentially co-localize with an FLC lacO transgenic reporter during prolonged cold, persisting after return to warm conditions. Our findings support a hybrid model of epigenetic memory in which nucleation of histone trimethylation is assisted by dynamic protein assemblies over extended durations. SlimVar therefore has potential to offer molecular insights into proteins expressed at physiological levels in a range of tissues.
... To further explore the observed structural changes, and to dissect the mechanism of fusion, we next performed single-vesicle imaging via a custom-built wide-field objective-type total internal reflection fluorescence microscope that enables the simultaneous imaging of DiI and DiD emission 42 . Here, POPC vesicles incorporating 2 % DiI were induced to fuse with LUVs incorporating 2 % DiD and 1 % biotin-PE such that fused species could be tethered to a glass coverslip functionalized with biotinylated BSA and NeutrAvidin (Figure 3a). ...
... Microfluidic flow cells were constructed as previously described 42 and coated with 0.1 mg/mL BSA-Biotin, 1 mg/mL BSA and 0.2 mg/mL NeutrAvidin. Biotinylated POPC vesicles incorporating 2 % DiD were mixed with non-biotinylated vesicles containing 2 % DiI (DiI-vesicle: DiD vesicle = 1: 3) at a final lipid concentration of 88 M in TX-100 rich media (50 mM Tris buffer, pH 8) prior to addition to the functionalized surface. ...
... Unbound vesicles were then removed by washing the flowcell with buffer. Objective-based TIRF microscopy was then performed on an inverted Nikon Eclipse Ti microscopy containing a 100 x NA 1.49 oil immersion objective lens (Nikon) 42 . Excitation was achieved from a continuous wave 532 nm laser line (Obis, Coherent) with the diameter of the illuminated region in the field of view calculated to be 26 m. ...
The structure, dynamics and function of lipid vesicles are heavily influenced by a range of physical forces, local microenvironmental effects and interactions with perturbative molecules, including detergents. Detergent-induced membrane interactions - critical for a wide range of applications including protein extraction and virus inactivation - varies in magnitude according to the detergent type and membrane composition, but the underlying mechanistic details remain largely under explored. Open questions relate to the precise molecular-level pathway of detergent-induced vesicle fusion, the nature of the fusion products, the influence of modulatory factors, and whether fusion states can be controllably harnessed for bionanotechnology. By using a lipid mixing assay based on Förster resonance energy transfer (FRET), and single-vesicle characterization approaches to assess vesicle heterogeneity, we identify that both freely-diffusing and surface-tethered sub-micron sized vesicles are induced to fuse by the widely used non-ionic detergent Triton-X 100. We demonstrate that the fusion process is a multi-step mechanism, characterized by discrete values of FRET efficiency between membrane-embedded donor and acceptor fluorophores, and involves vesicle docking, hemi-fusion and full lipid mixing, even at sub-solubilizing detergent concentrations. We present evidence that the fusion process is regulated by environmental factors including membrane composition and phase, and we dissect the kinetics of vesicle fusion in contact with solid surfaces using a label free quartz-crystal microbalance with dissipation monitoring approach. The presented strategies are likely to be applicable beyond the vesicle sizes and compositions studied here, and not only provide mechanistic insight into the multifaceted dynamics of vesicle fusion but also have implications for a wide range of biotechnological applications including drug delivery, sensor development, surfactant sensing, biomimetic formation, and microfluidics, where transport and manipulation of encapsulated cargo is essential.
... 284 In most applications, removal of photobleaching is desirable. However we note that in the case of single-molecule stepwise photobleaching analysis, where the stoichiometry of a complex containing labeled sub-units is estimated based on the number of photobleaching events, 285 both the labeling and detection efficiencies are critical for accurate estimations. 286 Single-molecule fluorescence methods are increasingly used across the chemical, biological, and medical sciences, and a growing interest is developing in near-infrared fluorophores as imaging probes, in part because they enable deeper imaging through organic material compared with shorter wavelength probes but also because they generally facilitate higher signal-to-noise ratios due to lower autofluorescence at longer wavelengths. ...
The explosive growth of single-molecule techniques is transforming our understanding of biology, helping to develop new physics inspired by emergent biological processes, and leading to emerging areas of nanotechnology. Key biological and chemical processes can now be probed with new levels of detail, one molecule at a time, from the nanoscopic dynamics of nature's molecular machines to an ever-expanding range of exciting applications across multiple length and time scales. Their common feature is an ability to render the underlying distribution of molecular properties that ensemble averaging masks and to reveal new insights into complex systems containing spatial and temporal heterogeneity. Small fluorescent probes are among the most adaptable and versatile for single-molecule sensing applications because they provide high signal-to-noise ratios combined with excellent specificity of labeling when chemically attached to target biomolecules or embedded within a host material. In this review, we examine recent advances in probe designs, their utility, and applications and provide a practical guide to their use, focusing on the single-molecule detection of nucleic acids, proteins, carbohydrates, and membrane dynamics. We also present key challenges that must be overcome to perform successful single-molecule experiments, including probe conjugation strategies, identify tradeoffs and limitations for each probe design, showcase emerging applications, and discuss exciting future directions for the community.
... An optimised angle of excitation beam delivery distinguishes PaTCH imaging from the traditionally used TIRF and HILO microscope settings, thereby facilitating the imaging of transmembrane proteins in mammalian cells 35 . In this study the angle of incidence of the excitation beam was set to a sub-critical angle of 55° by translation of a telescope lens, as calibrated using the lateral displacement of the beam downstream of the focal plane 56 . At this angle, we estimated that ~30% of the incident light is coupled into a reflected TIRF mode, with the associated enhancement of the excitation field at the surface. ...
The ability of tumors to establish a pro-tumorigenic microenvironment is an important point of investigation in the search for new therapeutics. Tumors form microenvironments in part by the “education” of immune cells attracted via chemotactic axes such as that of CCR5-CCL5. Further, CCR5 upregulation by cancer cells, coupled with its association with pro-tumorigenic features such as drug-resistance and metastasis, has suggested CCR5 as a therapeutic target. However, with several conformational “pools” being reported, phenotypic investigations must be capable of unveiling conformational heterogeneity. Addressing this challenge, we performed super-resolution structured illumination microscopy (SIM) and single molecule partially TIRF coupled HILO (PaTCH) microscopy of CCR5 in fixed cells. SIM data revealed a non-random spatial distribution of CCR5 assemblies, whilst Intensity-tracking of CCR5 assemblies from PaTCH images indicated dimeric sub-units independent of CCL5 perturbation. These biophysical methods can provide important insights into the structure and function of onco-immunogenic receptors and many other biomolecules.
... Microfluidic flow cells were constructed as described previously (Dresser et al., 2021) and sequentially incubated with 0.1 mg/ml BSA-Biotin, 1 mg/ml BSA, and 0.2 mg/ml NeutrAvidin. After each incubation step, the flow cells were rinsed with buffer (50 mM Tris, pH 8) to remove unbound material. ...
Lipid vesicles are valuable mesoscale molecular confinement vessels for studying membrane mechanics and lipid–protein interactions, and they have found utility among bio-inspired technologies, including drug delivery vehicles. While vesicle morphology can be modified by changing the lipid composition and introducing fusion or pore-forming proteins and detergents, the influence of extramembrane crowding on vesicle morphology has remained under-explored owing to a lack of experimental tools capable of capturing morphological changes on the nanoscale. Here, we use biocompatible polymers to simulate molecular crowding in vitro, and through combinations of FRET spectroscopy, lifetime analysis, dynamic light scattering, and single-vesicle imaging, we characterize how crowding regulates vesicle morphology. We show that both freely diffusing and surface-tethered vesicles fluorescently tagged with the DiI and DiD FRET pair undergo compaction in response to modest concentrations of sorbitol, polyethylene glycol, and Ficoll. A striking observation is that sorbitol results in irreversible compaction, whereas the influence of high molecular weight PEG-based crowders was found to be reversible. Regulation of molecular crowding allows for precise control of the vesicle architecture in vitro, with vast implications for drug delivery and vesicle trafficking systems. Furthermore, our observations of vesicle compaction may also serve to act as a mechanosensitive readout of extramembrane crowding.
... Total internal reflection fluorescence microscopy: labeled α-Syn imaging. The dual-labeled α-Syn samples were diluted to nanomolar concentrations to facilitate single-molecule imaging 63 . Samples were then added to a cover slide on the inside of the chamber and incubated for at least 30 minutes before being washed with 20-nm filtered buffer (20 mM Tris and 100 mM NaCl, pH 7.1). ...
Aggregation of alpha-synuclein (α-Syn) drives Parkinson’s disease (PD), although the initial stages of self-assembly and structural conversion have not been directly observed inside neurons. In this study, we tracked the intracellular conformational states of α-Syn using a single-molecule Förster resonance energy transfer (smFRET) biosensor, and we show here that α-Syn converts from a monomeric state into two distinct oligomeric states in neurons in a concentration-dependent and sequence-specific manner. Three-dimensional FRET-correlative light and electron microscopy (FRET-CLEM) revealed that intracellular seeding events occur preferentially on membrane surfaces, especially at mitochondrial membranes. The mitochondrial lipid cardiolipin triggers rapid oligomerization of A53T α-Syn, and cardiolipin is sequestered within aggregating lipid–protein complexes. Mitochondrial aggregates impair complex I activity and increase mitochondrial reactive oxygen species (ROS) generation, which accelerates the oligomerization of A53T α-Syn and causes permeabilization of mitochondrial membranes and cell death. These processes were also observed in induced pluripotent stem cell (iPSC)–derived neurons harboring A53T mutations from patients with PD. Our study highlights a mechanism of de novo α-Syn oligomerization at mitochondrial membranes and subsequent neuronal toxicity. This study tracked the initial self-assembly, oligomerization and structural conversion of α-synuclein inside neurons. Early seeding events occur on mitochondrial membranes, where oligomerization induces mitochondrial dysfunction and neuronal loss.
... Therefore, direct measurement of the number of fluorophore-tagged monomers present in a biomolecular assembly can be achieved by counting the total number of photobleaching steps in the fluorescence intensity profile over time (Leake et al., 2006;Lenn et al., 2011). This tool is well suited to determine stoichiometry of protein complex in living cells (Mehta et al., 2013;Hummert et al., 2021) as well as detect amyloidogenic protein oligomers (Dresser et al., 2021), although sometimes it is hampered by fast photobleaching, which limits the total number of photons that can be detected thereby giving a short time profile for analysis, or photo-induced blinking of the fluorophore (Dickson et al., 1997). ...
Aggregation kinetics of proteins and peptides have been studied extensively due to their significance in many human diseases, including neurodegenerative disorders, and the roles they play in some key physiological processes. However, most of these studies have been performed as bulk measurements using Thioflavin T or other fluorescence turn-on reagents as indicators of fibrillization. Such techniques are highly successful in making inferences about the nucleation and growth mechanism of fibrils, yet cannot directly measure assembly reactions at low protein concentrations which is the case for amyloid-β (Aβ) peptide under physiological conditions. In particular, the evolution from monomer to low-order oligomer in early stages of aggregation cannot be detected. Single-molecule methods allow direct access to such fundamental information. We developed a high-throughput protocol for single-molecule photobleaching experiments using an automated fluorescence microscope. Stepwise photobleaching analysis of the time profiles of individual foci allowed us to determine stoichiometry of protein oligomers and probe protein aggregation kinetics. Furthermore, we investigated the potential application of supervised machine learning with support vector machines (SVMs) as well as multilayer perceptron (MLP) artificial neural networks to classify bleaching traces into stoichiometric categories based on an ensemble of measurable quantities derivable from individual traces. Both SVM and MLP models achieved a comparable accuracy of more than 80% against simulated traces up to 19-mer, although MLP offered considerable speed advantages, thus making it suitable for application to high-throughput experimental data. We used our high-throughput method to study the aggregation of Aβ40 in the presence of metal ions and the aggregation of α-synuclein in the presence of gold nanoparticles.
... To further explore the structural changes, we performed single-vesicle FRET imaging by using a wide-field, objectivebased total internal reflection fluorescence microscope that enables the parallel imaging of DiI and DiD emission. 28,47 Here, the mean FRET efficiencies of single surfaceimmobilized vesicles labeled with 0.1% DiI and 0.1% DiD were monitored upon addition of Tween-20. Surface immobilization was achieved by incorporating 1% biotinylated lipids into the LUVs for coupling to a glass coverslip via biotin−NeutrAvidin interactions ( Figure 3A). ...
The solubilization of lipid membranes by Tween-20 is crucial for a number of biotechnological applications, but the mechanistic details remain elusive. Evidence from ensemble assays supports a solubilization model that encompasses surfactant association with the membrane and the release of mixed micelles to solution, but whether this process also involves intermediate transitions between regimes is unanswered. In search of mechanistic origins, increasing focus is placed on identifying Tween-20 interactions with controllable membrane mimetics. Here, we employed ultrasensitive biosensing approaches, including single-vesicle spectroscopy based on fluorescence and energy transfer from membrane-encapsulated molecules, to interrogate interactions between Tween-20 and submicrometer-sized vesicles below the optical diffraction limit. We discovered that Tween-20, even at concentrations below the critical micellar concentration, triggers stepwise and phase-dependent structural remodeling events, including permeabilization and swelling, in both freely diffusing and surface-tethered vesicles, highlighting the substantial impact the surfactant has on vesicle conformation and stability prior to lysis.
... Microfluidic flow cells were constructed as described previously 38 and sequentially incubated with 0.1 mg/mL BSA-Biotin, 1 mg/mL BSA and 0.2 mg/mL NeutrAvidin. After each incubation step the flow cells were rinsed with buffer (50 mM Tris, pH 8) to remove unbound material. ...
Lipid vesicles are valuable mesoscale molecular confinement vessels for studying membrane mechanics and lipid-protein interactions, and they have found utility among bio-inspired technologies including drug delivery vehicles. While vesicle morphology can be modified by changing the lipid composition and introducing fusion or pore-forming proteins and detergents, the influence of extramembrane crowding on vesicle morphology has remained under explored owing to a lack of experimental tools capable of capturing morphological changes on the nanoscale. Here, we use biocompatible polymers to simulate molecular crowding in vitro, and through combinations of FRET spectroscopy, lifetime analysis, dynamic light scattering and single-vesicle imaging, we characterize how crowding regulates vesicle morphology. We show that both freely-diffusing and surface-tethered vesicles fluorescently tagged with the DiI and DiD FRET pair undergo compaction in response to modest concentrations of sorbitol, polyethylene glycol and Ficoll. A striking observation is that sorbitol results in irreversible compaction, whereas the influence of high molecular weight PEG-based crowders was found to be reversible. Regulation of molecular crowding allows for precise control of vesicle architecture in vitro, with vast implications for drug delivery and vesicle trafficking systems. Furthermore, our observations of vesicle compaction may also serve to act as a mechanosensitive readout of extramembrane crowding.
