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Exploring the surface-capturing and releasing processes of nanocargo on the living cell membrane is critical for understanding the membrane translocation process. In this work, we achieve total internal reflection scattering (TIRS) illumination on a commercial dark-field optical microscope without the introduction of any additional optical components. By gradually reducing the diaphragm size in the excitation light path, the angle of the incident beam can be well manipulated. Under optimal conditions, the excitation light can be totally reflected at the glass/water interface, resulting in a thin layer of evanescent field for TIRS illumination. Due to the exponential decay feature of the evanescent field, the displacement of the nanocargo along the vertical direction can be directly resolved in the intensity track. With this method, we selectively monitor the dynamics of the transferrin-modified nanocargo on the living cell membrane. Transition between confined diffusion and long-range searching is involved in the binding site recognition process, which exhibits non-Gaussian and nonergodic-like behavior. More interestingly, 2D fast sliding and 3D hopping motions are also distinguished on the fluidic cell membrane, which is essentially modulated by the strength of ligand-receptor interactions, as revealed by the free-energy profiles. These heterogeneous and dynamic interactions together control the diffusion mode of the nanocargo on the lipid membrane and, thus, determine the cellular translocation efficiency.
Assuntos
Microscopia , Ligantes , Membrana Celular/metabolismoRESUMO
Bimetallic nanostructures are a promising candidate for plasmon-driven photocatalysis. However, knowledge on the generation and utilization of hot carriers in bimetallic nanostructures is still limited. In this work, we explored Pt position-dependent photocatalytic properties of bimetallic Au nanobipyramids (Au NBPs) with single-molecule fluorescence imaging. Compared with all-deposited core-shell nanostructures (aPt-Au NBPs), single-molecule imaging and simulation results show that the end-deposited bimetallic nanostructures (ePt-Au NBPs) can maintain a strong electromagnetic (EM) field and further promote the generation and transfer of energetic hot electrons for photocatalysis. Even though the Pt lattice is more stable than Au, the strong EM field at the sharp tips can boost lattice vibration, where enhanced spontaneous surface restructuring for active reaction site generation takes place. Significantly enhanced catalytic efficiency from ePt-Au NBPs is observed in contrast to that of Au NBPs and aPt-Au NBPs. These microscopic evidences offer valuable guidelines to design plasmon-based photocatalysts, particularly for bimetallic nanostructures.
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Anti-Aß therapy has dominated clinical trials for the prevention and treatment of Alzheimer's disease (AD). However, suppressing Aß aggregation and disintegrating mature fibrils simultaneously remains a great challenge. In this work, we developed a new strategy using a charged tubular supramolecule (CTS) with pillar[5]arene as the backbone and modifying amino and carboxyl groups at the tubular terminals (noted as CTS-A, CTS-A/C, and CTS-C, respectively) to suppress Aß fibrillation for the first time. According to the spectroscopic and microscopic characterizations, Aß40 fibrillation can be efficiently suppressed by CTS-A in a very low inhibitor:peptide (I:P) molar ratio (1:10). A greatly alleviated cytotoxic effect of Aß peptides after the inhibition or disaggregation process is further disclosed. The well-organized supramolecular structure drives multivalent interaction and gains enhanced efficiency on amyloid fibrillar modulation. These results open a new path for the design of supramolecules in the application of AD treatment.
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Doença de Alzheimer , Peptídeos beta-Amiloides , Amiloide/química , Peptídeos beta-Amiloides/química , Humanos , Fragmentos de PeptídeosRESUMO
Fluorescent chemosensors are powerful imaging tools in the fields of life sciences and engineering. Based on the principle of supramolecular chemistry, indicator displacement assay (IDA) provides an alternative approach for constructing and optimizing chemosensors, which has the advantages of simplicity, tunability, and modularity. However, the application of IDA in bioimaging continues to face a series of challenges, including interfering signals, background noise, and inconsistent spatial location. Accordingly, we herein report a supramolecular bioimaging strategy of Förster resonance energy transfer (FRET)-assisted IDA by employing macrocyclic amphiphiles as the operating platform. By merging FRET with IDA, the limitations of IDA in bioimaging were addressed. As a proof of concept, the study achieved mitochondria-targeted imaging of adenosine triphosphate in live cells with signal amplification. This study opens a non-covalent avenue for bioimaging with advancements in tunability, generality, and simplicity, apart from the covalent approach.
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Transferência Ressonante de Energia de Fluorescência , Corantes Fluorescentes/química , Indicadores e Reagentes/química , Células Hep G2 , Humanos , Substâncias Macromoleculares/análise , Espectrometria de FluorescênciaRESUMO
Artificial aquaporins are synthetic molecules that mimic the structure and function of natural aquaporins (AQPs) in cell membranes. The development of artificial aquaporins would provide an alternative strategy for treatment of AQP-related diseases. In this report, an artificial aquaporin has been constructed from an amino-terminated tubular molecule, which operates in a unimolecular mechanism. The artificial channel can work in cell membranes with high water permeability and selectivity rivaling those of AQPs. Importantly, the channel can restore wound healing of the cells that contain function-lost AQPs.
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Aquaporinas/farmacologia , Cicatrização/efeitos dos fármacos , Aquaporinas/química , Células Hep G2 , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Humanos , Modelos Moleculares , Simulação de Dinâmica Molecular , Estrutura Molecular , Imagem Individual de MoléculaRESUMO
Plasmonic nanoparticles with special localized surface plasmon resonance (LSPR) characters have been widely applied for optical sensing of various targets. With the combination of single nanoparticle imaging techniques, dynamic information of reactions and biological processes is obtained, facilitating the deep understanding of their principle and design of outstanding nanomaterials. In this review, we summarize the recently adopted optical analysis of diverse analytes based on plasmonic nanoparticles both in homogeneous solution and at the single-nanoparticle level. A brief introduction of LSPR is first discussed. Colorimetric and fluorimetric homogeneous detection examples by using different sensing mechanisms and strategies are provided. Single plasmonic nanoparticle-based analysis is concluded in two aspects: visualization of chemical reactions and understanding of biological processes. The basic sensing mechanisms and performances of these systems are introduced. Finally, this review highlights the challenges and future trend of plasmonic nanoparticle-based optical analysis systems.
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Different from traditional ensemble measurement methods, single-particle tracking (SPT) is a powerful approach to study the distribution of dynamic processes in a complex environment, providing crucial information from individual objects. This Feature summarizes the optical microscopic techniques and data analysis methods for scattering-based SPT. Some essential SPT-based applications within the cell are also delineated.
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Contamination of foods and feeds by aflatoxins is a universal yet serious problem all over the world. Particularly, aflatoxin B1 (AFB1) is the most primary form and readily leads to terrible damages to human health. In this work, we construct a sensitive aptasensor based on single-particle detection (SPD) to analyze AFB1 in peanut samples with luminescence resonance energy transfer (LRET) between the aptamer-modified upconversion nanoparticles (UCNPs-aptamer) and gold nanoparticles (GNPs). The UCNP-aptamer plays as the luminescence donor, while GNP acts as the energy acceptor. In the absence of AFB1, GNPs would adsorb onto the surface of UCNPs-aptamer because of the association between aptamers and GNPs, leading to luminescence quenching. However, the luminescence of UCNPs-aptamer is recovered gradually in the presence of AFB1, because the aptamers possess stronger affinity toward AFB1 than GNPs. Through statistically counting the number of luminescent particles on the glass slide surface, the concentration of AFB1 in solution is accurately determined. The linear dynamic range for AFB1 detection is from 3.13 to 125.00 ng/mL. The limit-of-detection (LOD) is 0.17 ng/mL, which is much lower than the allowable concentration in foods. As a result, this method would provide promising application for the sensitive detection of AFB1 in foods and feeds, which might make a meaningful contribution to food safety and public health in the future.
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Aflatoxina B1/análise , Aptâmeros de Nucleotídeos/química , Análise de Alimentos/métodos , Contaminação de Alimentos/análise , Arachis/química , Transferência Ressonante de Energia de Fluorescência/métodos , Análise de Alimentos/instrumentação , Ouro/química , Limite de Detecção , Luminescência , Nanopartículas Metálicas/químicaRESUMO
ß-Amyloid peptide (Aß) aggregation is the essential hallmark of neurodegenerative disorders such as Alzheimer's disease. Efficient inhibitors are highly desired for the prevention of Aß assembly that has been considered as the primary therapeutic strategy for neurodegenerative diseases. Apart from this, visualization of the aggregates and morphology at high spatial resolution is widely considered of crucial significance on biological treatment. In this work, we have developed small-sized (with diameter of â¼4.7 nm) and positively charged fluorescent conjugated polymer nanoparticles (CPNPs) with strong inhibition effect on Aß1-40 peptides fibrillation. Interestingly, the CPNPs also possess excellent photophysical properties, including high photon counts, robust blinking, and repetitive fluorescence switching, that are especially suitable for localization-based super-resolution imaging. Spatial resolution of â¼20 nm for these blinking CPNPs is readily achieved. According to the optical microscopic results, it was found that binding of CPNPs to the terminal of seed fibrils can effectively inhibit the fibrillation process. Owing to these attractive biological and unique photophysical properties, the small-sized CPNPs show high potential in a variety of super-resolution based biological applications.
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Peptídeos beta-Amiloides/química , Amiloide/química , Corantes Fluorescentes/química , Microscopia de Fluorescência/métodos , Nanopartículas/química , Imagem Óptica/métodos , Fragmentos de Peptídeos/química , Polímeros/química , Animais , Fluorescência , Humanos , Células PC12 , RatosRESUMO
In a clinical assay, enzymes are essential biomarkers for human disease diagnosis. In this work, a spectral-resolved single-particle detection (SPD) method is introduced to quantify alkaline phosphatase (ALP) activity in human serum with a supraparticle (SP) based on MnO2-modified gold nanoparticle (denoted as GNP@MnO2 SP) as the probe. In the presence of ALP, 2-phospho-l-ascorbic acid trisodium salt can be hydrolyzed into l-ascorbic acid, which serves as a good reduction agent to trigger the decomposition of the MnO2 shell on the GNP surface. Given that a trace amount of ALP exists, noticeable scattering color change can be detected at the single-particle level due to the sensitive localized surface plasmon resonance (LSPR) effect from GNPs. With spectral-resolved dark-field optical microscopy, a linear dynamic range of 0.06 to 2.48 mU/mL ( R2 = 0.99) and a very low limit of detection of 5.8 µU/mL for the ALP assay are readily achieved, which is more sensitive over the methods based on ensemble sample measurement. As a consequence, this strategy opens a new avenue for the design of an ultrasensitive detection method for disease-correlated biomarker diagnosis in the future.
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Fosfatase Alcalina/sangue , Fosfatase Alcalina/metabolismo , Ressonância de Plasmônio de Superfície , Ouro/química , Humanos , Compostos de Manganês/química , Nanopartículas Metálicas/química , Óxidos/química , Tamanho da Partícula , Propriedades de SuperfícieRESUMO
Generally, the message elucidated by the conventional analytical methods overlooks the heterogeneity of single objects, where the behavior of individual molecules is shielded. With the advent of optical microscopy imaging techniques, it is possible to identify, visualize and track individual molecules or nanoparticles under a biological environment with high temporal and spatial resolution. In this work, we summarize the commonly adopted optical microscopy techniques for bio-analytical assays in living cells, including total internal reflection fluorescence microscopy (TIRFM), super-resolution optical microscopy (SRM), and dark-field optical microscopy (DFM). The basic principles of these methods and some recent interesting applications in molecular detection and single-particle tracking are introduced. Moreover, the development in high-dimensional optical microscopy to achieve three-dimensional (3-D) as well as sub-diffraction localization and tracking of biomolecules is also highlighted.
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Fenômenos Fisiológicos Celulares , Processamento de Imagem Assistida por Computador/métodos , Microscopia de Fluorescência/instrumentação , Microscopia de Fluorescência/métodos , Imagem Óptica/instrumentação , Imagem Óptica/métodos , HumanosRESUMO
Rapid quantification of permanganate (MnO4-) in aqueous solution with a convenient and sensitive single-particle-detection (SPD) method is demonstrated by dark-field optical microscopy. The design is based on the selective etching of the Ag shell of a glucose-protected GNPs@Ag nanoparticle by MnO4-. In the presence of MnO4-, a noticeable red-shift of localized surface-plasmon resonance (LSPR, from blue to green) together with a tremendous decrease in the extinction coefficient from individual GNPs@Ag nanoparticles is observed. MnO4- can then be quantified by calculating the ratio between the number of green and blue particles on the cover glass surface after the etching process. A linear dynamic range of 0-6 µM and a limit of detection (LOD) as low as 46 nM were readily achieved, which are much lower than those of spectroscopic measurements in bulk solution. In tap water, a comparable LOD (50 nM) and satisfactory recovery efficiency are demonstrated. As a consequence of these merits, the method demonstrated herein will find promising applications for the ultrasensitive detection of MnO4- under complex milieu in the future.
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In this work, we demonstrate a convenient yet sensitive color-coded single-particle detection method for the quantification of pyrophosphate (PPi) by using single gold nanoparticle (GNP) as the probe. The design is based on GNP-dependent catalytic deposition of Cu onto the surface of GNPs with reduced nicotinamide adenine dinucleotide (NADH). Without PPi, Cu2+ can be directly reduced to Cu0 through the gold-catalyzed oxidization of NADH. In the presence of PPi, the coating process is impeded due to the strong coordination capability of PPi with Cu2+. The selective coating of Cu shell onto the GNPs surface results in the extraordinary red-shift of localized surface plasmon resonance from individual GNPs. By quantitatively counting the fraction of yellow particles with color-coded dark-field optical microscopy, the trace amounts of PPi in solution can be accurately quantified. The limit-of-detection is as low as 1.49 nM with a linear dynamic range of 0-4.29 µM, which is much lower than the spectroscopic measurements in bulk solution. In artificial urine sample, good recovery efficiency was achieved. As a consequence, the method demonstrated herein will find promising applications for the ultrasensitive detection of target biomolecules under biological milieu in the future.
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In this work, we demonstrated a single molecule photobleaching-based strategy for the ultrasensitive detection of adenosine. A modified split aptamer was designed to specifically recognize individual adenosine molecules in solution. The specific binding of dye-labeled short strand DNA probes onto the elongated aptamer strand in the presence of adenosine resulted in a concentration-dependent self-aggregation process. The degree-of-aggregation (DOA) of the short DNA probes on the elongated aptamer strand could then be accurately determined based on the single molecule photobleaching measurement. Through statistically analyzing the DOA under different target concentrations, a well-defined curvilinear relationship between the DOA and target molecule concentration (e.g., adenosine) was established. The limit-of-detection (LOD) is down to 44.5 pM, which is lower than those recently reported results with fluorescence-based analysis. Owing to the high sensitivity and excellent selectivity, the sensing strategy described herein would find broad applications in biomolecule analysis under complicated surroundings.
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Adenosina/análise , Aptâmeros de Nucleotídeos/química , Técnicas Biossensoriais/métodos , Carbocianinas/química , Sondas de DNA/química , Corantes Fluorescentes/química , Fluorescência , Limite de Detecção , Imagem Óptica/métodos , FotodegradaçãoRESUMO
Exploring the diffusion dynamics of a viral capsid proteins (VCP)-functionalized nanocarrier on a living cell membrane could provide much kinetic information for the better understanding of their biological functionality. Gold nanoparticles are an excellent core material of nanocarriers because of the good biocompatibility as well as versatile surface chemistry. However, due to the strong scattering background from subcellular organelles, it is a grand challenge to selectively image an individual nanocarrier on a living cell membrane. In this work, we demonstrated a convenient strategy to effectively screen the scattering background from living cells for single-particle imaging with a polarization-resolved dual-channel imaging module. By taking advantage of the polarization of anisotropic gold nanoparticles (gold nanorods, GNRs), the signals from cell components could be counteracted after subtracting the sequential images one by one, while those transiently rotating GNRs on the cell membrane still exist in the processed image. In contrast to the previously reported methods, this method does not require a complicated optical setup alignment and sophisticated digital image analysis process. According to the single-particle imaging results, the majority of VCP-GNRs were anchoring on the cell membrane with confined diffusion. Interestingly, on further inspection of the diffusion trajectories, the particles displayed anomalous confined diffusion with randomly distributed large walking steps during the whole track. Non-Gaussian step distribution was noted, indicating heterogeneous binding and desorption processes on the cell membrane. As a consequence of the robust background screening capability, this approach would find broad applications for single-particle imaging under a noisy environment, e.g., living cells.
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Proteínas do Capsídeo/análise , Infecções por Circoviridae/virologia , Circovirus/química , Ouro/química , Hepatócitos/virologia , Nanopartículas Metálicas/química , Imagem Óptica/métodos , Anisotropia , Infecções por Circoviridae/patologia , Desenho de Equipamento , Células Hep G2 , Hepatócitos/patologia , Humanos , Microscopia/instrumentação , Microscopia/métodos , Imagem Óptica/instrumentaçãoRESUMO
Prostate-specific antigen (PSA) is an intercellular glycoprotein produced primarily by the prostate gland, which is commonly chosen as the initial target for the early diagnosis of prostate cancer. In this work, we demonstrate a simple yet sensitive sandwich-type single-particle enumeration (SPE) immunoassay for the quantitative detection of PSA in a flow chamber. The design is based on the luminescence resonance energy transfer (LRET) between upconversion nanoparticles (UCNPs) and gold nanoparticles (GNPs). The carboxyl group-functionalized UCNPs are conjugated with anti-PSA detection antibodies (Ab1) and serve as the luminescence energy donor, while GNPs are modified with anti-PSA capture antibodies (Ab2) and act as the energy acceptor. In the presence of target antigen (i.e., PSA), the specific immnuoreaction brings the donor and acceptor into close proximity, resulting in quenched luminescence. Through statistical counting of the target-dependent fluorescent particles on the glass slide surface, the quantity of antigens in the solution is accurately determined. The dynamic range for PSA detection in Tris-buffered saline (TBS) is 0-500 pM and the limit-of-detection (LOD) is 1.0 pM, which is much lower than the cutoff level in patients' serum samples. In the serum sample assay, comparable LOD was also achieved (i.e., 2.3 pM). As a consequence, this method will find promising applications for the selective detection of cancer biomarkers in clinical diagnosis.
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Biomarcadores Tumorais/análise , Ouro/química , Imunoensaio , Imunoadsorventes/química , Nanopartículas Metálicas/química , Antígeno Prostático Específico/análise , Humanos , Tamanho da Partícula , Propriedades de SuperfícieRESUMO
Recently, the development of new fluorescent probes for the ratiometric detection of target objects inside living cells has received great attention. Normally, the preparation, modification as well as conjugation procedures of these probes are complicated. On this basis, great efforts have been paid to establish convenient method for the preparation of dual emissive nanosensor. In this work, a functional dual emissive carbon dots (dCDs) was prepared by a one-pot hydrothermal carbonization method. The dCDs exhibits two distinctive fluorescence emission peaks at 440 and 624 nm with the excitation at 380 nm. Different from the commonly reported dCDs, this probe exhibited an interesting wavelength dependent dual responsive functionality toward lysine (440 nm) and pH (624 nm), enabling the ratiometric detection of these two targets. The quantitative analysis displayed that a linear range of 0.5-260 µM with a detection limit of 94 nM toward lysine and the differentiation of pH variation from 1.5 to 5.0 could be readily realized in a ratiometric strategy, which was not reported before with other carbon dots (CDs) as the probe. Furthermore, because of the low cytotoxicity, good optical and colloidal stability, and excellent wavelength dependent sensitivity and selectivity toward lysine and pH, this probe was successfully applied to monitor the dynamic variation of lysine and pH in cellular systems, demonstrating the promising applicability for biosensing in the future.
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Carbono/química , Lisina/análise , Pontos Quânticos/química , Fluorescência , Células HeLa , Humanos , Concentração de Íons de Hidrogênio , Estrutura Molecular , Imagem Óptica , Tamanho da Partícula , Propriedades de SuperfícieRESUMO
Understanding the detailed diffusion behavior of the nanocargo on lipid membrane can afford deep insight into the surface chemistry controlled translocation mechanism for the rational design of an efficient delivery system. By tracking the diffusion trajectory of transacting activator of transcription (TAT, a cell penetrating peptide) peptides-modified nanocargo on lipid membrane, bulk-mediated (intermittent hopping) diffusion was observed for the first time after a blended modification of TAT peptides and polyethylene glycol (PEG) molecules onto the nanoparticle surface. In contrast to random walk or confined diffusion, the nanoparticles could be temporarily confined for random waiting times between surface displacements produced by excursions through the bulk fluid, which was not noted before. Non-Gaussian distributed step length (with a stretched power law like tail) was observed, making large displacements much more probable than one would predict for regular Gaussian decay. This kind of larger displacement would therefore significantly facilitate a kinetically controlled surface searching process like heterogeneous penetration site recognition on a fluidic membrane with suitable spatial orientation.
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Peptídeos Penetradores de Células/metabolismo , Bicamadas Lipídicas/metabolismo , Nanopartículas Metálicas/química , Sequência de Aminoácidos , Peptídeos Penetradores de Células/química , Difusão , Ouro/química , HIV-1/metabolismo , Humanos , Bicamadas Lipídicas/química , Microscopia Eletrônica de Transmissão , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/metabolismo , Polietilenoglicóis/química , Produtos do Gene tat do Vírus da Imunodeficiência Humana/química , Produtos do Gene tat do Vírus da Imunodeficiência Humana/metabolismoRESUMO
In this work, a hybridized nanoparticle with fluorescence/dark-field dual-modality imaging capability was prepared by nanoprecipitation of fluorescent conjugated polymer onto the surface of silica-coated rod-shape plasmonic nanoparticle. According to the spectroscopic and microscopic characterizations, the fluorescence intensity of conjugated polymer poly[(9,9-dioctylfluorenyl-2,7-diyl)-alt-co-(1,4-benzo(2,1',3)-thiadiazole)] (PFBT) could be enhanced around 2-fold after assembling onto the silica-decorated metal nanorod surface compared with the fluorescence intensity of regular PFBT polymer dots without the metal core. The in situ nanorod etching experiment further confirmed this result at the single particle level. In addition to the fluorescence enhancement effect, improved fluorescence stability was obtained from the single particle fluorescence intensity characterizations. As a consequence, this self-assembled functional nanoparticle could be extensively applied to biological imaging such as cellular labeling and single particle tracking owing to the novel and unique optical features, for example, the superior optical stability and specific identification capability from the scattering and fluorescence domain, respectively. Furthermore, the amendable peripheral polymer surface of this nanostructure will promote its applications in biological sensing and imaging-guided functional molecule delivery in the future.
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Understanding the mechanistic information on many kinetic processes requires the exploration of dynamic rotational information on the target object at the single particle (or molecule) level. In this work, we developed a new strategy, total internal reflection scattering (TIRS) microscopy, to determine the full three-dimensional (3D) angular information on a single gold nanorod (GNR) close to the liquid/solid interface. It was found that the 3D orientational information on individual GNR could be readily elucidated by using p-polarized TIRS illumination through deciphering the orientation-coded intensity distribution pattern in a single TIRS image. In comparison with the previously reported strategies, this method does not require complicated focal plane correction, affording a versatile pathway to track the rotational dynamics close to the interface in a high throughput manner. The methodology presented here, therefore, demonstrates a promising approach that can be applied to fluidic membranes, including membranes with polymers, bound proteins, and so on.