Search Results (54)

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

Background: In normal prostate cells, receptors for oxytocin (OT), a peptide involved in regulating prostate growth are sequestered within membrane microdomains called caveolae. During cancer progression, polymerase-transcript-release factor (PTRF) is downregulated, caveolae structures are lost and receptors move onto the cell membrane. This study investigated whether proteins responsible for caveolae formation were affected by the OT peptide, also, how OT treatment affected oxytocin receptor (OTR) movement within living cells. Methods: Normal human prostate epithelial cells (PrEC) expressing caveolin and PTRF and androgen-independent (PC3) cancer cells expressing caveolin but not PTRF were used. OTR, caveolin and PTRF expression was determined in human prostate tissue. Results: PTRF expression decreased in tissue alongside an increase in malignancy. Caveolin-1 expression was downregulated by OT treatment. Caveolin-2 was decreased by OT in PrEC cells but increased in PC3 cells. PTRF was decreased by OT in PrEC. TIRF microscopy showed OTR translocated from caveolae to caveolae in normal cells, whereas OTR moved without restraint in malignant cells, possibly stimulating signaling pathways. Conclusions: This study provides evidence for the ability of OT to regulate caveolin and PTRF expression. This study elucidates possible mechanisms by which cell receptors and caveolae proteins interact to enhance cell proliferation. Full article

Background/Objectives: This study evaluated the appropriateness of transmucosal immediate-release fentanyl (TIRF) prescriptions in a Madrid emergency room during 2019 and 2022, following a 2018 warning about off-label use. Methods: TIRF prescription in the emergency room search yielded 993 patients in 2019 and 1499 in 2022, of which 140 were randomized for the study, 70 in 2019, and 70 in 2022. Dose appropriateness and indication for TIRF were analyzed according to established criteria. Results: Despite a high prevalence of cancer diagnoses (77.9%, 109/140), only 32.9% (46/140) of patients met the appropriateness criteria pre-hospitalization. This improved to 42.5% (51/120) at discharge, but the change was not statistically significant overall. However, focusing on surviving patients reveals a significant improvement in appropriateness, increasing from 30.83% (37/120) to 42.50% (p = 0.002). This improvement was particularly pronounced in 2022 (p = 0.0269), but not in 2019 (p = 0.0771). Interestingly, appropriateness in patients with prior TIRF prescriptions remained relatively stable from pre-hospitalization (46.75%) to discharge (48.78%). A concerningly high proportion of patients with cancer diagnoses (68.75%) received low-dose opioid therapy (<60 MME) at discharge, and 36.8% of patients over 80 years old were co-prescribed benzodiazepines, contradicting prescribing guidelines. Conclusions: This study found inappropriate TIRF prescriptions were common in an emergency room setting, often due to low pre-hospital opioid doses. While hospitalization improved TIRF appropriateness in survivors, especially in 2022, concerning prescribing practices persisted. This emphasizes the need for better education and interventions to ensure safe and effective TIRF use. Full article

Tropomyosins (Tpms) are rod-shaped proteins that interact head-to-tail to form a continuous polymer along both sides of most cellular actin filaments. Head-to-tail interaction between adjacent Tpm molecules and the formation of an overlap complex between them leads to the assembly of actin filaments with one type of Tpm isoform in time and space. Variations in the affinity of tropomyosin isoforms for different actin structures are proposed as a potential sorting mechanism. However, the detailed mechanisms of the spatio-temporal sorting of Tpms remain elusive. In this study, we investigated the early intermediates during actin–tropomyosin filament assembly, using a skeletal/cardiac Tpm isoform (Tpm1.1) and a cytoskeletal isoform (Tpm1.6) that differ only in the last 27 amino acids. We investigated how the muscle isoform Tpm1.1 and the cytoskeletal isoform Tpm1.6 nucleate domains on the actin filament, and tested whether (1) recruitment is affected by the actin isoform (muscle vs. cytoskeletal) and (2) whether there is specificity in recruiting the same isoform to a domain at these early stages. To address these questions, actin filaments were exposed to low concentrations of fluorescent tropomyosins in solution. The filaments were immobilized onto glass coverslips and the pattern of decoration was visualized by TIRF microscopy. We show that at the early assembly stage, tropomyosins formed multiple distinct fluorescent domains (here termed “cluster”) on the actin filaments. An automated image analysis algorithm was developed and validated to identify clusters and estimate the number of tropomyosins in each cluster. The analysis showed that tropomyosin isoform sorting onto an actin filament is unlikely to be driven by a preference for nucleating on the corresponding muscle or cytoskeletal actin isoforms, but rather is facilitated by a higher probability of incorporating the same tropomyosin isoforms into an early assembly intermediate. We showed that the 27 amino acids at the end of each tropomyosin seem to provide enough molecular information for the attachment of the same tropomyosin isoforms adjacent to each other on an actin filament. This results in the formation of homogeneous clusters composed of the same isoform rather than clusters with mixed isoforms. Full article

In the field of studies on the “Neural Synapses” in the nervous system, its experts manually (or pseudo-automatically) detect the bio-molecule clusters (e.g., of proteins) in many TIRF (Total Internal Reflection Fluorescence) images of a fluorescent cell and analyze their static/dynamic behaviors. This paper proposes a novel method for the automatic detection of the bio-molecule clusters in a TIRF image of a fluorescent cell and conducts several experiments on its performance, e.g., mAP @ IoU (mean Average Precision @ Intersection over Union) and F1-score @ IoU, as an objective/quantitative means of evaluation. As a result, the best of the proposed methods achieved 0.695 as its mAP @ IoU = 0.5 and 0.250 as its F1-score @ IoU = 0.5 and would have to be improved, especially with respect to its recall @ IoU. But, the proposed method could automatically detect bio-molecule clusters that are not only circular and not always uniform in size, and it can output various histograms and heatmaps for novel deeper analyses of the automatically detected bio-molecule clusters, while the particles detected by the Mosaic Particle Tracker 2D/3D, which is one of the most conventional methods for experts, can be only circular and uniform in size. In addition, this paper defines and validates a novel similarity of automatically detected bio-molecule clusters between fluorescent cells, i.e., SimMolCC, and also shows some examples of SimMolCC-based applications. 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

Many kinds of spatio-temporal data in our daily lives, such as the trajectory data of moving objects, stream natively. Streaming systems exhibit significant advantages in processing streaming data due to their distributed architecture, high throughput, and real-time performance. The use of streaming processing techniques for spatio-temporal data applications is a promising research direction. However, due to the strong dynamic nature of data in streaming processing systems, traditional spatio-temporal indexing techniques based on relatively static data cannot be used directly in stream-processing environments. It is necessary to study and design new spatio-temporal indexing strategies. Hence, we propose a workload-controllable dynamic spatio-temporal index based on the R-tree. In order to restrict memory usage, we formulate an replace into and batch-REMOVE (I&BR) method and append a collection mechanism to the traditional R-tree. To improve the updating performance, we propose a time-identified R-tree (TIR). Moreover, we propose a distributed system prototype called a time-identified R-tree farm (TIRF). Experiments show that the TIR could work in a scenario with a controllable usage of memory and a stable response time. The throughput of the TIRF could reach 1 million points per second. The performance of a range search in the TIRF is many times better than in PostgreSQL, which is a widely used database system for spatio-temporal applications. 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

SIDT2 is a lysosomal protein involved in the degradation of nucleic acids and the transport of cholesterol between membranes. Previous studies identified two “cholesterol recognition/interaction amino acid consensus” (CRAC) motifs in SIDT1 and SIDT2 members. We have previously shown that the first CRAC motif (CRAC-1) is essential for protein translocation to the PM upon cholesterol depletion in the cell. In the present study, we show that SIDT2 and the apolipoprotein A1 (ApoA1) form a complex which requires the second CRAC-2 motif in SIDT2 to be established. The overexpression of SIDT2 and ApoA1 results in enhanced ApoA1 secretion by HepG2 cells. This is not observed when overexpressing the SIDT2 with the CRAC-2 domain mutated to render it unfunctional. All these results provide evidence of a novel role for SIDT2 as a protein forming a complex with ApoA1 and enhancing its secretion to the extracellular space. 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

The mechanical properties of living cells, including their shape, rigidity, and internal dynamics play a crucial role in their physiology and pathology. Still, the relations between the physiological cell state and its rigidity and surface vibrations remain poorly understood. Here, we have employed AFM measurements on T cells and found a negative relation between cell surface stiffness and its vibrations. Blocking T-type Ca⁺⁺-channels using Mibefradil reduced cortical actin tension in these cells and enhanced their membrane vibrations and dissipation of intracellular mechanical work to the cell surroundings. We also found increased vibrations of cell membranes in five different malignant cells lines derived from T cell leukemia, lung, prostate, bladder, and melanoma cancers, as compared to their corresponding benign cells. This was demonstrated by utilizing TIRF microscopy in single cells and dynamic laser speckles measurements in an in vitro model of multiple cells in a tissue. Our results show that cell membrane vibrations and dissipation of mechanical work are higher in malignant cells relative to benign cells. Accordingly, these properties may be used to detect and monitor cellular and tissue malignancies. Full article

The synaptic protein–DNA complexes, formed by specialized proteins that bridge two or more distant sites on DNA, are critically involved in various genetic processes. However, the molecular mechanism by which the protein searches for these sites and how it brings them together is not well understood. Our previous studies directly visualized search pathways used by SfiI, and we identified two pathways, DNA threading and site-bound transfer pathways, specific to the site-search process for synaptic DNA–protein systems. To investigate the molecular mechanism behind these site-search pathways, we assembled complexes of SfiI with various DNA substrates corresponding to different transient states and measured their stability using a single-molecule fluorescence approach. These assemblies corresponded to specific–specific (synaptic), non-specific–non-specific (non-specific), and specific–non-specific (pre-synaptic) SfiI–DNA states. Unexpectedly, an elevated stability in pre-synaptic complexes assembled with specific and non-specific DNA substrates was found. To explain these surprising observations, a theoretical approach that describes the assembly of these complexes and compares the predictions with the experiment was developed. The theory explains this effect by utilizing entropic arguments, according to which, after the partial dissociation, the non-specific DNA template has multiple possibilities of rebinding, effectively increasing the stability. Such difference in the stabilities of SfiI complexes with specific and non-specific DNA explains the utilization of threading and site-bound transfer pathways in the search process of synaptic protein–DNA complexes discovered in the time-lapse AFM experiments. 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

Evanescent field excitation is a powerful means to achieve a high surface-to-bulk signal ratio for bioimaging and sensing applications. However, standard evanescent wave techniques such as TIRF and SNOM require complex microscopy setups. Additionally, the precise positioning of the source relative to the analytes of interest is required, as the evanescent wave is critically distance-dependent. In this work, we present a detailed investigation of evanescent field excitation of near-surface waveguides written using femtosecond laser in glass. We studied the waveguide-to-surface distance and refractive index change to attain a high coupling efficiency between evanescent waves and organic fluorophores. First, our study demonstrated a reduction in sensing efficiency for waveguides written at their minimum distance to the surface without ablation as the refractive index contrast of the waveguide increased. While this result was anticipated, it had not been previously demonstrated in the literature. Moreover, we found that fluorescence excitation by waveguides can be enhanced using plasmonic silver nanoparticles. The nanoparticles were also organized in linear assemblies, perpendicular to the waveguide, with a wrinkled PDMS stamp technique, which resulted in an excitation enhancement of over 20 times compared to the setup without nanoparticles. Full article