Total Internal Reflection Fluorescence Imaging Research Articles

Simultaneous TIRF imaging of subplasmalemmal Ca2+ dynamics and granule fusions in insulin-secreting INS-1 cells reveals coexistent synchronized and asynchronous release

The basal and glucose-induced insulin secretion from pancreatic beta cells is a tightly regulated process that is triggered in a Ca2+-dependent fashion and further positively modulated by substances that raise intracellular levels of adenosine 3′,5′-cyclic monophosphate (cAMP) or by certain antidiabetic drugs. In a previous study, we have temporally resolved the subplasmalemmal [Ca2+]i dynamics in beta cells that are characterized by trains of sharply delimited spikes, reaching peak values up to 5 µM. Applying total internal reflection fluorescence (TIRF) microscopy and synaptopHluorin to visualize fusion events of individual granules, we found that several fusion events can coincide within 50 to 150 ms. To test whether subplasmalemmal [Ca2+]i microdomains around single or clustered Ca2+ channels may cause a synchronized release of insulin-containing vesicles, we applied simultaneous dual-color TIRF microscopy and monitored Ca2+ fluctuations and exocytotic events in INS-1 cells at high frame rates. The results indicate that fusions can be triggered by subplasmalemmal Ca2+ spiking. This, however, does account for a minority of fusion events. About 90 %-95 % of fusion events either happen between Ca2+ spikes or incidentally overlap with subplasmalemmal Ca2+ spikes. We conclude that only a fraction of exocytotic events in glucose-induced and tolbutamide- or forskolin-enhanced insulin release from INS-1 cells is tightly coupled to Ca2+ microdomains around voltage-gated Ca2+ channels.

Deep Learning Promotes Profiling of Multiple miRNAs in Single Extracellular Vesicles for Cancer Diagnosis.

Extracellular vesicle microRNAs (EV miRNAs) are critical noninvasive biomarkers for early cancer diagnosis. However, accurate cancer diagnosis based on bulk analysis is hindered by the heterogeneity among EVs. Herein, we report an approach for profiling single-EV multi-miRNA signatures by combining total internal reflection fluorescence (TIRF) imaging with a deep learning (DL) algorithm for the first time. This innovative technique allows for the precise characterization of EV miRNAs at the single-vesicle level, overcoming the challenges posed by EV heterogeneity. TIRF with high resolution and a signal-to-noise ratio can simultaneously detect multi-miRNAs in situ in individual EVs. DL algorithm avoids complicated and inaccurate artificial feature extraction, achieving automated high-resolution image analysis. Using this approach, we reveal that the main variation of EVs from 5 cancer cells and normal plasma is the triple-positive EV subpopulation, and the classification accuracy of single triple-positive EVs from 6 sources can reach above 95%. In the clinical cohort, 20 patients (5 lung cancer, 5 breast cancer, 5 cervical cancer, and 5 colon cancer) and 5 healthy controls are predicted with an overall accuracy of 100%. This single-EV strategy provides new opportunities for exploring more specific EV biomarkers to achieve cancer diagnosis and classification.

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.

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.

Trends in Single-Molecule Total Internal Reflection Fluorescence Imaging and Their Biological Applications with Lab-on-a-Chip Technology.

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.

Super-resolved total internal reflection fluorescence microscopy using random illuminations

A benefit of random illumination microscopy (RIM) is that it improves the resolution and linearity of the brightness of structured illumination microscopy using minimally controlled speckled illumination. Here, we implemented RIM in the total internal reflection fluorescence (TIRF) configuration for imaging biological processes close to the coverslip surface. Using standard TIRF objectives, we separated fluorescent lines 60 nm apart and achieved high contrast 86 nm resolution on fixed biological samples. Applied to live macrophages, TIRF-RIM provided two-color dynamic images of paxillin nanoclusters with remarkable spatial (96–120 nm) and temporal (1–8 Hz) resolutions, respectively. The simple experimental setup and imaging protocol together with the robustness of the data processing to leaks and aberrations make TIRF-RIM a method of choice for super-resolution TIRF imaging.

Indicator-dependent differences in detection of local intracellular Ca2+ release events evoked by voltage-gated Ca2+ entry in pancreatic β-cells

Genetically encoded Ca2+ indicators have become widely used in cell signalling studies as they offer advantages over cell-loaded dye indicators in enabling specific cellular or subcellular targeting. Comparing responses from dye and protein-based indicators may provide information about indicator properties and cell physiology, but side-by-side recordings in cells are scarce. In this study, we compared cytoplasmic Ca2+ concentration ([Ca2+]i) changes in insulin-secreting β-cells recorded with commonly used dyes and indicators based on circularly permuted fluorescent proteins. Total internal reflection fluorescence (TIRF) imaging of K+ depolarization-triggered submembrane [Ca2+]i increases showed that the dyes Fluo-4 and Fluo-5F mainly reported stable [Ca2+]i elevations, whereas the proteins R-GECO1 and GCaMP5G more often reported distinct [Ca2+]i spikes from an elevated level. [Ca2+]i spiking occurred also in glucose-stimulated cells. The spikes reflected Ca2+ release from the endoplasmic reticulum, triggered by autocrine activation of purinergic receptors after exocytotic release of ATP and/or ADP, and the spikes were consequently prevented by SERCA inhibition or P2Y1-receptor antagonism. Widefield imaging, which monitors the entire cytoplasm, increased the spike detection by the Ca2+ dyes. The indicator-dependent response patterns were unrelated to Ca2+ binding affinity, buffering and mobility, and probably reflects the much slower dissociation kinetics of protein compared to dye indicators. Ca2+ dyes thus report signalling within the submembrane space excited by TIRF illumination, whereas the protein indicators also catch Ca2+ events originating outside this volume. The study highlights that voltage-dependent Ca2+ entry in β-cells is tightly linked to local intracellular Ca2+ release mediated via an autocrine route that may be more important than previously reported direct Ca2+ effects on phospholipase C or on intracellular channels mediating calcium-induced calcium release.

Molecular Basis for Actin Polymerization Kinetics Modulated by Solution Crowding.

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.

Biphasic reinforcement of nascent adhesions by vinculin.

Vinculin is an integral component of integrin adhesions, where it functions as a molecular clutch coupling intracellular contraction to the extracellular matrix. Quantitating its contribution to the reinforcement of newly forming adhesions, however, requires ultrasensitive cell force assays covering short time and low force ranges. Here, we have combined atomic force microscopy-based single-cell force spectroscopy (SCFS) and optical tweezers force spectroscopy to investigate the role of vinculin in reinforcement of individual nascent adhesions during the first 5 min of cell contact with fibronectin or vitronectin. At minimal adhesion times (5-10 s), mouse embryonic fibroblast (MEF) wildtype (wt) and vinculin knock-out (vin(-/-) ) cells develop comparable adhesion forces on the scale of several individual integrin-ligand bonds, confirming that vinculin is dispensable for adhesion initiation. In contrast, after 60 to 120 s, adhesion strength and traction reinforce quickly in wt cells, while remaining low in vin(-/-) cells. Re-expression of full-length vinculin or a constitutively active vinculin mutant (vinT12) in MEF vin(-/-) cells restored adhesion and traction with the same efficiency, while vinculin with a mutated talin-binding head region (vinA50I) or missing the actin-binding tail-domain (vin880) was ineffective. Integrating total internal reflection fluorescence imaging into the SCFS setup furthermore enabled us to correlate vinculin-green fluorescent protein (GFP) recruitment to nascent adhesion sites with the built-up of vinculin-dependent adhesion forces directly. Vinculin recruitment and cell adhesion reinforcement followed synchronous biphasic patterns, suggesting vinculin recruitment, but not activation, as the rate-limiting step for adhesion reinforcement. Combining sensitive SCFS with fluorescence microscopy thus provides insight into the temporal sequence of vinculin-dependent mechanical reinforcement in nascent integrin adhesions.

Abstract 5875: Rab37 mediates intracellular trafficking and membrane presentation of PD-1 in T cells to foster an immunosuppressive microenvironment in lung cancer

Abstract Programmed cell death protein 1 (PD-1) expressed on the surface of CD8+ T cells is known as an immune checkpoint protein. High expression of PD-1 leads to T-cell dysfunction in the tumor microenvironment (TME). Our published studies show that Rab37 small GTPase-mediated vesicular trafficking of cell surface proteins and cytokines is critical in immunosuppressive TME. Here, we identify novel mechanisms of intracellular trafficking and plasma membrane presentation of PD-1 mediated by Rab37. Confocal immunofluorescence (IF) and vesicles isolation data demonstrated that PD-1 colocalized with Rab37-specific vesicles in T cells in a GTP-dependent manner. Total internal reflection fluorescence imaging, membrane fractionation, and flow cytometric analyses confirmed the dynamic trafficking and membrane presentation of PD-1 by Rab37. In addition, sucrose density gradient centrifugation and IF revealed that the level of glycosylated wild-type PD-1 in membrane compartment was more than glycosylation mutant PD-1, suggesting that glycosylation mutant PD-1 delayed in recruitment to the Rab37 vesicles and thus stalled in membrane presentation. Furthermore, T cell proliferation and activity were downregulated in Rab37 wild-type splenocytes co-cultured with cancer cells compared to those from Rab37 knockout mice using various T cell functional assays. Clinically, the multiplex IF-immunohistochemical assay indicated that the tumor infiltrated Rab37+/PD1+/TIM-3+/CD8+ exhaustion T cells were more in late staged lung cancer patients compared to those in early staged patients. Importantly, patients with Rab37+/PD-1+/TIM-3+/CD8+ tumor infiltrated T cells expression profile correlated with poor overall survival. Our results provide first trafficking mode of PD-1 mediated by Rab37 small GTPase and novel evidence of the tumor promoting function of Rab37/PD-1 axis in T cells of the tumor microenvironment. Citation Format: Wan-Ting Kuo, I-Ying Kuo, Shih-Ting Wu, Wu-Chou Su, Yi-Ching Wang. Rab37 mediates intracellular trafficking and membrane presentation of PD-1 in T cells to foster an immunosuppressive microenvironment in lung cancer. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5875.

MesoTIRF: A prism-based Total Internal Reflection Fluorescence illuminator for high resolution, high contrast imaging of large cell populations

Total Internal Reflection Fluorescence (TIRF) illumination bypasses the axial diffraction limit of light by using an evanescent field to excite fluorophores close to a sample substrate. However, standard TIRF imaging through the objective requires a high numerical aperture (NA) to generate the evanescent wave. Available lenses have a high magnification with a correspondingly small field of view—ranging from ∼50 μm to 1 mm in diameter. Switching to the older prism-TIRF configuration introduced by Axelrod in the 1980s might seem to remove the requirement for high objective NA and allow the use of existing large-field objectives. Unfortunately, these lenses are unsuitable because their throughput of light is too low for TIRF imaging. As such, high sensitivity TIRF imaging over a much larger mesoscopic field has yet to be demonstrated. We have developed a prism-based TIRF illuminator for the Mesolens—a highly corrected objective lens with an unparalleled ratio of NA to magnification. The imaging field of the Mesolens is 204 times larger than that of the TIRF objectives previously described, increasing the optical throughput of the optical system by a factor of 25 compared to an off-the-shelf microscope objective of the same magnification. We demonstrate MesoTIRF imaging of cell specimens and show the multi-wavelength capability of the modality across more than 700 cells in a single image.

Conformational dynamics of glucagon receptor domains revealed by single-molecule fluorescence microscopy.

G protein-coupled receptors (GPCRs) are integral membrane proteins involved in many cellular signaling processes. Representing the largest family of proteins with approximately 800 members in humans, GPCRs are the target for more than 40% of current pharmaceutical drugs. The structural and dynamic information is vital to understanding the activation mechanism of these receptors. Despite the availability of high-resolution structures of GPCRs at different conformational states, the dynamics of those conformational states at the molecular level are missing. We used total internal reflection fluorescence (TIRF) imaging to investigate the conformational dynamics of the glucagon receptor (GCGR), a class B family GPCR involved in glucose homeostasis. We expressed and purified GCGR from mammalian cells and analyzed them at a single molecule level. We used single-molecule Förster resonance energy transfer (smFRET) to observe the conformational dynamics of the donor and acceptor fluorophore-labeled GCGR molecules. We observed ligand-dependent slow and reversible conformational fluctuation of N-terminal extra cellular domain (ECD) without ligand binding. The addition of a saturated concentration of peptide ligand (Glucagon) altered the dynamics towards the lower FRET states. These results provide new insight into the structural dynamics of glucagon receptor and receptor activation.

Hyperstabilization of T cell microvilli contacts by chimeric antigen receptors.

T cells typically recognize their ligands using a defined cell biology-the scanning of their membrane microvilli (MV) to palpate their environment-while that same membrane scaffolds T cell receptors (TCRs) that can signal upon ligand binding. Chimeric antigen receptors (CARs) present both a therapeutic promise and a tractable means to study the interplay between receptor affinity, MV dynamics and T cell function. CARs are often built using single-chain variable fragments (scFvs) with far greater affinity than that of natural TCRs. We used high-resolution lattice lightsheet (LLS) and total internal reflection fluorescence (TIRF) imaging to visualize MV scanning in the context of variations in CAR design. This demonstrated that conventional CARs hyper-stabilized microvillar contacts relative to TCRs. Reducing receptor affinity, antigen density, and/or multiplicity of receptor binding sites normalized microvillar dynamics and synapse resolution, and effector functions improved with reduced affinity and/or antigen density, highlighting the importance of understanding the underlying cell biology when designing receptors for optimal antigen engagement.

Combined Scattering, Interferometric, and Fluorescence Oblique Illumination for Live Cell Nanoscale Imaging

To determine the molecular and/or mechanical basis of cell migration using live cell imaging tools, it is necessary to correlate multiple 3D spatiotemporal events simultaneously. Fluorescence nanoscopy and label-free nanoscale imaging can complement each other by providing both molecular specificity and structural dynamics of sub-cellular structures. A combined imaging system would permit obtaining quantitative 3D spatial temporal details of individual cellular components. In this paper, we empirically determined optimal azimuthal scanning angles of rotating beams to achieve simultaneous and label-free nanoscale and fluorescence imaging. Label-free nanoscale imaging here refers to interferometric, bright-field (BF) and dark-field (DF) rotating coherence scattering (ROCS) microscopy, while fluorescence refers to high-inclined laminated oblique (HiLO) and total internal reflection fluorescence (TIRF) imaging. The combined capabilities of interferometric, scattering, and fluorescence imaging enable (1) the identification of molecular targets (substrate or organelle), (2) quantification of 3D cell morphodynamics, and (3) tracking of intracellular organelles in 3D. This combined imaging tool was then used to characterize migrating platelets and endothelial cells, both critical to the process of infection and wound healing. The combined imaging results of more than ∼1000 platelets suggest that serum albumin (bovine) is necessary for platelets to migrate and scavenge fibrin/fibrinogen. Furthermore, we identified new asynchronous membrane fluctuations between the leading and rear edges of a migrating platelet. We further demonstrated that interferometric imaging permits the quantification of mitochondrial dynamics on human lung microvascular endothelial cells. Our data suggests that axial displacement of mitochondria is minimal when it is closer to the nucleus or the leading edge of a cell membrane. Taken together, this combined nanoscopy platform helps to quantify multiple spatial temporal events of a migrating cell that will undoubtedly open ways to new quantitative correlative nanoscale live cell imaging.

Decreased FAK activity and focal adhesion dynamics impair proper neurite formation of medium spiny neurons in Huntington's disease.

Huntington's disease (HD) is a neurodegenerative disorder caused by a polyglutamine expansion in the protein huntingtin (HTT) [55]. While the final pathological consequence of HD is the neuronal cell death in the striatum region of the brain, it is still unclear how mutant HTT (mHTT) causes synaptic dysfunctions at the early stage and during the progression of HD. Here, we discovered that the basal activity of focal adhesion kinase (FAK) is severely reduced in a striatal HD cell line, a mouse model of HD, and the human post-mortem brains of HD patients. In addition, we observed with a FRET-based FAK biosensor [59] that neurotransmitter-induced FAK activation is decreased in HD striatal neurons. Total internal reflection fluorescence (TIRF) imaging revealed that the reduced FAK activity causes the impairment of focal adhesion (FA) dynamics, which further leads to the defect in filopodial dynamics causing the abnormally increased number of immature neurites in HD striatal neurons. Therefore, our results suggest that the decreased FAK and FA dynamics in HD impair the proper formation of neurites, which is crucial for normal synaptic functions [52]. We further investigated the molecular mechanism of FAK inhibition in HD and surprisingly discovered that mHTT strongly associates with phosphatidylinositol 4,5-biphosphate, altering its normal distribution at the plasma membrane, which is crucial for FAK activation [14, 60]. Therefore, our results provide a novel molecular mechanism of FAK inhibition in HD along with its pathological mechanism for synaptic dysfunctions during the progression of HD.

Single Small Extracellular Vesicle (sEV) Quantification by Upconversion Nanoparticles.

Cancer-derived small extracellular vesicles (sEVs) are potential circulating biomarkers in liquid biopsies. However, their small sizes, low abundance, and heterogeneity in molecular makeups pose major technical challenges for detecting and characterizing them quantitatively. Here, we demonstrate a single-sEV enumeration platform using lanthanide-doped upconversion nanoparticles (UCNPs). Taking advantage of the unique optical properties of UCNPs and the background-eliminating property of total internal reflection fluorescence (TIRF) imaging technique, a single-sEV assay recorded a limit of detection 1.8 × 106 EVs/mL, which was nearly 3 orders of magnitude lower than the standard enzyme-linked immunosorbent assay (ELISA). Its specificity was validated by the difference between EpCAM-positive and EpCAM-negative sEVs. The accuracy of the UCNP-based single-sEV assay was benchmarked with immunomagnetic-beads flow cytometry, showing a high correlation (R2> 0.99). The platform is suitable for evaluating the heterogeneous antigen expression of sEV and can be easily adapted for biomarker discoveries and disease diagnosis.

Aging Alters Integrin‐mediated Vascular Smooth Muscle Function in Soleus Feed Arteries

IntroductionPrevious data have shown that aging impairs vasomotor function and alters vascular smooth muscle cell (VSMC) properties. Integrins play an important role in regulating many cellular functions including arterial contractility. However, how aging affects integrin function, and their contribution to impaired constriction in aged arteries has not yet been elucidated. Thus, the purpose of this study was to investigate the age‐induced alteration of integrin function in isolated VSMC and in intact soleus muscle resistance arteries.MethodsSoleus muscle feed arteries (SFA) were isolated from young (4 mo) and old (24 mo) male Fischer 344 rats, cannulated with glass micropipettes and pressurized at 90cm H2O. Endothelial cells were removed (denuded) by passing 5 ml of air through the artery lumen. Vasoconstrictor responses were assessed by adding of norepinephrine (NE; 10‐9‐10‐4M), angiotensin II (Ang II; 10‐11‐10‐7 M), or phenylephrine (PE; 10‐9‐10‐4 M) in the presence or absence of RGD, an integrin inhibitory peptide. RGE, a non‐inhibitory peptide was used as control. To investigate changes in integrin expression and recruitment at focal adhesions with age, RT‐qPCR, cell adhesion assays, and total internal reflection fluorescence (TIRF) imaging were performed using VSMC isolated from young and old SFA.ResultsOld denuded SFA had reduced vasoconstrictor responses to NE, PE, and Ang II. In the presence of the RGD‐inhibitory peptide, constrictor responses to Ang II were significantly inhibited in denuded old vs. young SFA. Constrictor responses to NE and PE were not significantly altered by RGD. Constrictor responses to NE, PE, and Ang II were also not affected by RGE. Gene expression of specific integrins expressed by VSMC were downregulated in old VSMC compared to young. In addition, RGD‐treated VSMC showed decreased adhesion to fibronectin in both young and old VSMC compared to RGE non‐inhibitory peptide. Quantitative analysis of TIRF images showed that recruitment of integrin α5 and β3 was diminished in old VSMC while β1 was not altered with age.ConclusionsTaken together, these data suggest that aging leads to integrin dysfunction, which contributes to decreased contractile properties of vascular smooth muscle in SFA.

Signal Transduction Mechanisms Quantitatively Observed One Molecule at a Time

Improved single-molecule methods can largely increase our understanding of underlying molecular mechanism during cellular signal transduction. In contrast to conventional bulk methods, monitoring molecules one at a time can circumvent averaging effects and acquire unique information. With single-molecule techniques, quantitative characterizations can be achieved at microscopic level, especially for biochemical systems with strong heterogeneity. Here we review four fundamental single-molecule techniques including total internal reflection fluorescence imaging, single-molecule fluorescence recovery after photobleaching, single-molecule Förster resonance energy transfer, and fluorescence correlation/cross-correlation spectroscopy. These techniques are frequently employed in quantitatively investigating the molecular translocation, protein-protein interactions, aggregations, and conformational dynamics involved in the signal transduction both in vitro and in vivo. We also summarized the basic principles and implementations of these single-molecule techniques, as well as the conjunct applications extending the single-molecule measurements to multiple dimensions.

A2A adenosine receptor conformational dynamics revealed by single-molecule fluorescence

G protein-coupled receptors (GPCRs) are integral membrane proteins involved in a wide range of cellular signaling processes. The structural and dynamic information is important in understanding the activation mechanism and essential for designing new drugs. However, despite the availability of high-resolution structures at different conformational states, the dynamics of those conformational states at the molecular level are still missing. Here, we used total internal reflection fluorescence (TIRF) imaging to investigate the conformational dynamics of the human A2A adenosine receptor (A2AAR), a representative class A GPCR, at the single-molecule level.

An economic, square-shaped flat-field illumination module for TIRF-based super-resolution microscopy

Super-resolution microscopy allows complex biological assemblies to be observed with remarkable resolution. However, the presence of uneven Gaussian-shaped illumination hinders its use in quantitative imaging or high-throughput assays. Methods developed to circumvent this problem are often expensive, hard to implement, or not applicable to total internal reflection fluorescence imaging. We herein demonstrate a cost-effective method to overcome these challenges using a small square-core multimodal optical fiber as the coupler. We characterize our method with synthetic, recombinant, and cellular systems imaged under total internal reflection fluorescence and highly inclined and laminated optical sheet illuminations to demonstrate its ability to produce highly uniform images under all conditions.