Total Internal Reflection Fluorescence Research Articles

Observation of reactions in single molecules/nanoparticles using light microscopy

AbstractRecent techniques for direct observation of single molecules or nanoparticles provide methodologies for imaging the activation sites of heterogeneous catalysts (spatially resolved) and observing intermediates that are not visible in the ensemble average (temporally resolved). Accordingly, the primary challenge for related experiments is obtaining sufficient spatial and temporal resolutions for microscopic observation of the chemical reaction of interest. This review discusses recent advances in fluorescence—for example, total internal reflection fluorescence (TIRF)—and dark‐field microscopy—for example, imaging plasmonic probes—used for observing organic, inorganic, and biological reactions. The following key factors for microscopic observation of chemical reactions are discussed: (1) design of the chemical reaction and probe, (2) selection of microscope based on reaction's temporal information, and (3) use of machine learning algorithms to analyze the sequence imaging data. This review summarizes experimental techniques and detailed examples of reactions at the single molecule and nanoparticle level. Furthermore, it discusses avenues of development. These observations can guide the development of new and systematic methodological approaches for investigating important unsolved problems in chemistry.

Phosphoserine-86-HSPB1 (pS86-HSPB1) is cytoplasmic and highly induced in rat myometrium at labour

Uterine myocytes during pregnancy proceed through a series of adaptations and collectively transform into a powerfully contractile tissue by term. Previous work has indicated that members of the heat shock protein (HSP) B family of stress proteins are associated with the process of adaptation and transformation. Utilizing immunoblot analyses, widefield epifluorescence and total internal reflection (TIRF) microscopy, this study investigated the temporal and spatial detection of HSPB1 phosphorylated on serine-86 (pS86-HSPB1) in rat myometrium during pregnancy, the role of uterine distension in regulation of pS86-HSPB1, and the comparative localization with pS15-HSPB1 in rat myometrial tissue as well as in an immortalized human myometrial cell line. Immunoblot detection of pS86-HSPB1 was significantly elevated during late pregnancy and labour. In particular, pS86-HSPB1 was significantly increased at day (d)22 and d23 (labour) compared with all other timepoints assessed. Localization of pS86-HSPB1 in myometrium became prominent at d22 and d23 with cytoplasmic detection around myometrial cell nuclei. Furthermore, pS86-HSPB1 detection was found to be significantly elevated in the gravid rat uterine myometrium compared with the non-gravid tissue at d19 and d23. Both widefield epifluorescence and TIRF microscopy examination of human myometrial cells demonstrated that pS15-HSPB1 was prominently localized to focal adhesions, while pS82-HSPB1 (homologous to rodent pS86-HSPB1) was primarily located in the cell cytoplasm. Our data demonstrate that levels of phosphorylated HSPB1 increase just prior to and during labour, and that uterine distension is a stress-inducing signal for HSPB1 phosphorylation. The exact roles of these phosphorylated forms in myometrial cells remain to be determined.

Enhancing flotation separation of fine copper oxide from silica by microbubble assisted hydrophobic aggregation

One of the main challenges facing the flotation of base metal oxide minerals is their fine sizes of particles as a result of fine grinding to achieve a desired degree of liberation for low-grade and finely disseminated complex ores. This study aims at studying hydrophobic aggregation and in situ gas nucleation of fine copper oxides with sodium dodecyl sulfate (SDS) as a selective collector to enhance CuO recovery. Experimental results by in situ and real time particle size measurement using focused beam reflectance measurement (FBRM) coupled with particle vision measurement (PVM) showed an enhanced aggregation of fine CuO by SDS adsorption. Particle aggregation assisted by gas nuclei which were generated in situ on CuO by high intensity agitation (HIA) was visualized by total internal reflection fluorescence microscopy (TIRFM). It is the enhanced aggregation with nanobubbles as bridges that improved its recovery from fine silica. Coupled with the calculation using the extended DLVO theory, contact angle and zeta potential of fine particles were measured to understand the critical role of hydrophobic forces in aggregation of fine CuO particles. The copper ions dissolved from the fine copper oxide were found to inadvertently activate fine silica flotation which could be effectively depressed by ethylene diamine tetraacetic acid (EDTA). A combination of SDS with EDTA was proposed and demonstrated to achieve selective recovery of fine copper oxide from fine silica gangue. The results from this study provide directions for enhancing copper recovery from low grade and finely disseminated copper oxidize ores, in particular from tenorite ores.

Probing Transient Riboswitch Structures via Single Molecule Accessibility Analysis.

Riboswitches are a class of RNA motifs in the untranslated regions of bacterial messenger RNAs (mRNAs) that can adopt different conformations to regulate gene expression. The binding of specific small molecule or ion ligands, or other RNAs, influences the conformation the riboswitch adopts. Single Molecule Kinetic Analysis of RNA Transient Structure (SiM-KARTS) offers an approach for probing this structural isomerization, or conformational switching, at the level of single mRNA molecules. SiM-KARTS utilizes fluorescently labeled, short, sequence-complementary DNA or RNA oligonucleotide probes that transiently access a specific RNA conformation over another. Binding and dissociation to a surface-immobilized target RNA of arbitrary length are monitored by Total Internal Reflection Fluorescence Microscopy (TIRFM) and quantitatively analyzed, via spike train and burst detection, to elucidate the rate constants of isomerization, revealing mechanistic insights into riboswitching.

Insights into the Effect of the Adsorption Preference of Additives on the Anisotropic Growth of ZSM-5 Zeolite.

Zeolite morphology plays a crucial role in affecting catalytic performance, while is persistently challenging to tailor through crystal anisotropic growth. It has been recognized that specific additives can be introduced into the synthesis of zeolites to achieve anisotropic growth, however their role and the underlying mechanism are not well understood. Herein, the effect of eight specific additives on the anisotropic growth of the ZSM-5 zeolite is unveiled within the framework of crystallization engineering. Either an inhibition effect or a promotion effect is revealed for each additive according to the crystallization kinetics. The adsorption preference of typical additives on different surfaces was demonstrated by total internal reflection fluorescence microscopy (TIRFM) and transmission X-ray microscopy (TXM) together with 3D reconstruction. The calculated adsorption energy difference between MFI [100]/[101] and [010] surfaces was proposed as a key descriptor to estimate the possible morphology induced by additive. ZSM-5 zeolites varying from sphere-like, plate-like to noodle-like morphology could be synthesized by employing specific additives with increasing adsorption strength difference on distinct surfaces.

The N-terminal disease–associated R5L Tau mutation increases microtubule shrinkage rate due to disruption of microtubule-bound Tau patches

Regulation of the neuronal microtubule cytoskeleton is achieved through the coordination of microtubule-associated proteins (MAPs). MAP-Tau, the most abundant MAP in the axon, functions to modulate motor motility, participate in signaling cascades, as well as directly mediate microtubule dynamics. Tau misregulation is associated with a class of neurodegenerative diseases, known as tauopathies, including progressive supranuclear palsy, Pick's disease, and Alzheimer's disease. Many disease-associated mutations in Tau are found in the C-terminal microtubule-binding domain. These mutations decrease microtubule-binding affinity and are proposed to reduce microtubule stability, leading to disease. N-terminal disease-associated mutations also exist, but the mechanistic details of their downstream effects are not as clear. Here, we investigate the effect of the progressive supranuclear palsy–associated N-terminal R5L mutation on Tau-mediated microtubule dynamics using an in vitro reconstituted system. We show that the R5L mutation does not alter Tau interactions with tubulin by fluorescence correlation spectroscopy. Using total internal reflection fluorescence microscopy, we determined that the R5L mutation has no effect on microtubule growth rate, catastrophe frequency, or rescue frequency. Rather, the R5L mutation increases microtubule shrinkage rate. We determine this is due to disruption of Tau patches, larger order Tau complexes known to form on the GDP-microtubule lattice. Altogether, these results provide insight into the role of Tau patches in mediating microtubule dynamics and suggesting a novel mechanism by which mutations in the N-terminal projection domain reduce microtubule stability.

Imaging the Infection Cycle of T7 at the Single Virion Level.

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

A Clathrin light chain A reporter mouse for in vivo imaging of endocytosis.

Clathrin-mediated endocytosis (CME) is one of the best studied cellular uptake pathways and its contributions to nutrient uptake, receptor signaling, and maintenance of the lipid membrane homeostasis have been already elucidated. Today, we still have a lack of understanding how the different components of this pathway cooperate dynamically in vivo. Therefore, we generated a reporter mouse model for CME by fusing eGFP endogenously in frame to clathrin light chain a (Clta) to track endocytosis in living mice. The fusion protein is expressed in all tissues, but in a cell specific manner, and can be visualized using fluorescence microscopy. Recruitment to nanobeads recorded by TIRF microscopy validated the functionality of the Clta-eGFP reporter. With this reporter model we were able to track the dynamics of Alexa594-BSA uptake in kidneys of anesthetized mice using intravital 2-photon microscopy. This reporter mouse model is not only a suitable and powerful tool to track CME in vivo in genetic or disease mouse models it can also help to shed light into the differential roles of the two clathrin light chain isoforms in health and disease.

Kinesin-1, -2, and -3 motors use family-specific mechanochemical strategies to effectively compete with dynein during bidirectional transport.

Bidirectional cargo transport in neurons requires competing activity of motors from the kinesin-1, -2, and -3 superfamilies against cytoplasmic dynein-1. Previous studies demonstrated that when kinesin-1 attached to dynein-dynactin-BicD2 (DDB) complex, the tethered motors move slowly with a slight plus-end bias, suggesting kinesin-1 overpowers DDB but DDB generates a substantial hindering load. Compared to kinesin-1, motors from the kinesin-2 and -3 families display a higher sensitivity to load in single-molecule assays and are thus predicted to be overpowered by dynein complexes in cargo transport. To test this prediction, we used a DNA scaffold to pair DDB with members of the kinesin-1, -2, and -3 families to recreate bidirectional transport in vitro, and tracked the motor pairs using two-channel TIRF microscopy. Unexpectedly, we find that when both kinesin and dynein are engaged and stepping on the microtubule, kinesin-1, -2, and -3 motors are able to effectively withstand hindering loads generated by DDB. Stochastic stepping simulations reveal that kinesin-2 and -3 motors compensate for their faster detachment rates under load with faster reattachment kinetics. The similar performance between the three kinesin transport families highlights how motor kinetics play critical roles in balancing forces between kinesin and dynein, and emphasizes the importance of motor regulation by cargo adaptors, regulatory proteins, and the microtubule track for tuning the speed and directionality of cargo transport in cells.

I-scope: a compact automated fluorescence microscope for cell counting applications in low resource settings

Fluorescence microscopy has widespread applications across biological sciences. It has been routinely used for cell counting, which provides a preliminary diagnostic test for many infectious diseases. Conventional fluorescence microscopes are bulky, expensive, time-intensive and laborious. They often require trained operators to acquire and analyze data. We report a compact automated digital fluorescence microscopy system, i-scope, for cell counting applications. The i-scope employs a total internal reflection fluorescence (TIRF) mode of sample illumination, along with a brightfield mode. It has a magnification of 30X, an optical resolution of ∼0.2 μm/pixel and offers sample scanning over 20 mm × 20 mm. A custom-written program enables automated image acquisition and analysis, thereby enhancing ease of operation. It has a compact form-factor and has been developed into a standalone system with a processing unit, screen, and other accessories to offer a portable and economic point-of-care diagnostic solution in low-resource settings. We analysed the performance of the i-scope for milk somatic cell enumeration and benchmarked it against that of a conventional fluorescence microscope.

Improved immunoassay sensitivity and specificity using single-molecule colocalization

Enzyme-linked immunosorbent assays (ELISAs) are a cornerstone of modern molecular detection, but the technique still faces notable challenges. One of the biggest problems is discriminating true signal generated by target molecules versus non-specific background. Here, we developed a Single-Molecule Colocalization Assay (SiMCA) that overcomes this problem by employing total internal reflection fluorescence microscopy to quantify target proteins based on the colocalization of fluorescent signal from orthogonally labeled capture and detection antibodies. By specifically counting colocalized signals, we can eliminate the effects of background produced by non-specific binding of detection antibodies. Using TNF-α, we show that SiMCA achieves a three-fold lower limit of detection compared to conventional single-color assays and exhibits consistent performance for assays performed in complex specimens such as serum and blood. Our results help define the pernicious effects of non-specific background in immunoassays and demonstrate the diagnostic gains that can be achieved by eliminating those effects.

Gain-of-Function Dynamin-2 Mutations Linked to Centronuclear Myopathy Impair Ca2+-Induced Exocytosis in Human Myoblasts.

Gain-of-function mutations of dynamin-2, a mechano-GTPase that remodels membrane and actin filaments, cause centronuclear myopathy (CNM), a congenital disease that mainly affects skeletal muscle tissue. Among these mutations, the variants p.A618T and p.S619L lead to a gain of function and cause a severe neonatal phenotype. By using total internal reflection fluorescence microscopy (TIRFM) in immortalized human myoblasts expressing the pH-sensitive fluorescent protein (pHluorin) fused to the insulin-responsive aminopeptidase IRAP as a reporter of the GLUT4 vesicle trafficking, we measured single pHluorin signals to investigate how p.A618T and p.S619L mutations influence exocytosis. We show here that both dynamin-2 mutations significantly reduced the number and durations of pHluorin signals induced by 10 μM ionomycin, indicating that in addition to impairing exocytosis, they also affect the fusion pore dynamics. These mutations also disrupt the formation of actin filaments, a process that reportedly favors exocytosis. This altered exocytosis might importantly disturb the plasmalemma expression of functional proteins such as the glucose transporter GLUT4 in skeletal muscle cells, impacting the physiology of the skeletal muscle tissue and contributing to the CNM disease.

Microcontact Printing of Biomolecules on Various Polymeric Substrates: Limitations and Applicability for Fluorescence Microscopy and Subcellular Micropatterning Assays.

Polymeric materials play an emerging role in biosensing interfaces. Within this regard, polymers can serve as a superior surface for binding and printing of biomolecules. In this study, we characterized 11 different polymer foils [cyclic olefin polymer (COP), cyclic olefin copolymer (COC), polymethylmethacrylate (PMMA), DI-Acetate, Lumirror 4001, Melinex 506, Melinex ST 504, polyamide 6, polyethersulfone, polyether ether ketone, and polyimide] to test for the applicability for surface functionalization, biomolecule micropatterning, and fluorescence microscopy approaches. Pristine polymer foils were characterized via UV–vis spectroscopy. Functional groups were introduced by plasma activation and epoxysilane-coating. Polymer modification was evaluated by water contact angle measurement and X-ray photoelectron spectroscopy. Protein micropatterns were fabricated using microcontact printing. Functionalized substrates were characterized via fluorescence contrast measurements using epifluorescence and total internal reflection fluorescence microscopy. Results showed that all polymer substrates could be chemically modified with epoxide functional groups, as indicated by reduced water contact angles compared to untreated surfaces. However, transmission and refractive index measurements revealed differences in important optical parameters, which was further proved by fluorescence contrast measurements of printed biomolecules. COC, COP, and PMMA were identified as the most promising alternatives to commonly used glass coverslips, which also showed superior applicability in subcellular micropatterning experiments.

Inflammatory activation of the FcγR and IFNγR pathways co-influences the differentiation and activity of osteoclasts.

Osteoclasts are polykaryons formed by cell–cell fusion of highly motile progenitors of the myeloid lineage. Osteoclast activity can preserve skeletal strength and bone homeostasis. However, osteoclasts are responsible for bone destruction in rheumatoid arthritis (RA). Fc receptors activated by IgG immune complexes (IC) can boost osteoclast differentiation and bone loss in the course of RA. In contrast, interferon (IFN) γ secreted by immune cells blocks osteoclast activation. Despite their hypothetical importance in the regulation of osteoclast differentiation in RA, the interconnection between the two pathways has not been described so far. Here, we show by total internal reflection fluorescence (TIRF) microscopy that FcγR3 and IFNγ receptor (IFNγR) locate at close vicinity to each other on the human osteoclast surface. Moreover, the average distance increases during the differentiation process. Interestingly, FcγR and IFNγR activation shapes the position of both receptors to each other. Surprisingly, the inhibitory action of IFNγ on in-vitro human osteoclast differentiation depends on the osteoclast differentiation stage. Indeed, IFNγR activation in early osteoclast precursors completely inhibits the formation of polynucleated osteoclasts, while in premature osteoclasts, it further enhanced their fusion. In addition, gene expression analyses showed that IFNγR activation on early precursor cells but not on premature osteoclasts could induce FcγR expression, suggesting a co-regulation of both receptors on human osteoclast precursors. Phosphokinase array data of precursor cells demonstrate that the observed divergence of IFNγR signaling is dependent on the mitogen−activated protein kinase (MAPK) downstream signaling pathway. Overall, our data indicate that FcγR and IFNγR signaling pathways co-influence the differentiation and activity of osteoclasts dependent on the differentiation state, which might reflect the different stages in RA.

Single molecule microscopy reveals diverse actions of substrate sequences that impair ClpX AAA+ ATPase function

AAA+ (ATPases Associated with diverse cellular Activities) proteases unfold substrate proteins by pulling the substrate polypeptide through a narrow pore. To overcome the barrier to unfolding, substrates may require extended association with the ATPase. Failed unfolding attempts can lead to a slip of grip, which may result in substrate dissociation, but how substrate sequence affects slippage is unresolved. Here, we measured single molecule dwell time using total internal reflection fluorescence microscopy, scoring time-dependent dissociation of engaged substrates from bacterial AAA+ ATPase unfoldase/translocase ClpX. Substrates comprising a stable domain resistant to unfolding and a C-terminal unstructured tail, tagged with a degron for initiating translocase insertion, were used to determine dwell time in relation to tail length and composition. We found greater tail length promoted substrate retention during futile unfolding. Additionally, we tested two tail compositions known to frustrate unfolding. A poly-glycine tract (polyG) promoted release, but only when adjacent to the folded domain, whereas glycine-alanine repeats (GAr) did not promote release. A high complexity motif containing polar and charged residues also promoted release. We further investigated the impact of these and related motifs on substrate degradation rates and ATP consumption, using the unfoldase–protease complex ClpXP. Here, substrate domain stability modulates the effects of substrate tail sequences. polyG and GAr are both inhibitory for unfolding, but act in different ways. GAr motifs only negatively affected degradation of highly stable substrates, which is accompanied by reduced ClpXP ATPase activity. Together, our results specify substrate characteristics that affect unfolding and degradation by ClpXP.

The changing view of insulin granule mobility: From conveyor belt to signaling hub.

Before the advent of TIRF microscopy the fate of the insulin granule prior to secretion was deduced from biochemical investigations, electron microscopy and electrophysiological measurements. Since Calcium-triggered granule fusion is indisputably necessary to release insulin into the extracellular space, much effort was directed to the measure this event at the single granule level. This has also been the major application of the TIRF microscopy of the pancreatic beta cell when it became available about 20 years ago. To better understand the metabolic modulation of secretion, we were interested to characterize the entirety of the insulin granules which are localized in the vicinity of the plasma membrane to identify the characteristics which predispose to fusion. In this review we concentrate on how the description of granule mobility in the submembrane space has evolved as a result of progress in methodology. The granules are in a state of constant turnover with widely different periods of residence in this space. While granule fusion is associated +with prolonged residence and decreased lateral mobility, these characteristics may not only result from binding to the plasma membrane but also from binding to the cortical actin web, which is present in the immediate submembrane space. While granule age as such affects granule mobility and fusion probability, the preceding functional states of the beta cell leave their mark on these parameters, too. In summary, the submembrane granules form a highly dynamic heterogeneous population and contribute to the metabolic memory of the beta cells.

Asphaltene precipitation under controlled mixing conditions in a microchamber

Solvent exchange is a controlled process for dilution-induced phase separation. This work utilizes the solvent exchange method to reveal the effect of the mixing dynamics on the asphaltene precipitation process under 20 different mixing conditions using a model system of n-heptane and asphaltene in toluene. The external mixing between the asphaltene solution and the paraffinic solvent is strictly controlled. We employed high-spatial resolution total internal reflection fluorescence microscope to detect asphaltene precipitates with a resolution up to ∼ 200 nm. A multiphysics model is used to simulate the evolution of oversaturation pulse in the solvent exchange process. Based on the simulation results, we predicted the effect of the flow rate, dimension, orientation of the microfluidic chamber, and temperature on the surface coverage and size distribution of asphaltene precipitates. The model predictions of all factors corroborate with the experimental observations. Local concentration of the solvent and shear forces are found to be the two main reasons for the change of asphaltene precipitation caused by mixing dynamics. However, the influence of thermodynamics is more critical than the mixing dynamics as temperature changes. Through a combination of experimental and simulation studies, this work illuminates the significance of the transportation process for the final morphology of asphaltene precipitates and provides a in-depth insight into the mechanism of mixing dynamics on the asphaltene precipitation. A smart mixing may be to boost new phase formation without excessive solvent consumption.

Femtomolar detection of SARS-CoV-2 via peptide beacons integrated on a miniaturized TIRF microscope.

The novel coronavirus SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) continues to pose a substantial global health threat. Along with vaccines and targeted therapeutics, there is a critical need for rapid diagnostic solutions. In this work, we use computational protein modeling tools to suggest molecular beacon architectures that function as conformational switches for high-sensitivity detection of the SARS-CoV-2 spike protein receptor binding domain (S-RBD). Integrating these beacons on a miniaturized total internal reflection fluorescence (mini-TIRF) microscope, we detect the S-RBD and pseudotyped SARS-CoV-2 with limits of detection in the femtomolar range. We envision that our designed mini-TIRF platform will serve as a robust platform for point-of-care diagnostics for SARS-CoV-2 and future emergent viral threats.

Complexin-1 and synaptotagmin-1 compete for binding sites on membranes containing PtdInsP2

Complexin-1 is an essential protein for neuronal exocytosis that acts to depress spontaneous fusion events while enhancing evoked neurotransmitter release. In addition to binding soluble N-ethylmaleimide-sensitive factor attachment protein receptors, it is well established that complexin associates with membranes in a manner that depends upon membrane curvature. In the present work, we examine the membrane binding of complexin using electron paramagnetic resonance spectroscopy, fluorescence anisotropy, and total internal reflection fluorescence microscopy. The apparent membrane affinity of complexin is found to strongly depend upon the concentration of protein used in the binding assay, and this is a result of a limited number of binding sites for complexin on the membrane interface. Although both the N- and C-terminal regions of complexin associate with the membrane interface, membrane affinity is driven by its C-terminus. Complexin prefers to bind liquid-disordered membrane phases and shows an enhanced affinity toward membranes containing phosphatidylinositol 4-5-bisphosphate (PI(4,5)P2). In the presence of PI(4,5)P2, complexin is displaced from the membrane surface by proteins that bind to or sequester PI(4,5)P2. In particular, the neuronal calcium sensor synaptotagmin-1 displaces complexin from the membrane but only when PI(4,5)P2 is present. Complexin and synaptotagmin compete on the membrane interface in the presence of PI(4,5)P2, and this interaction may play a role in calcium-triggered exocytosis by displacing complexin from its fusion-inhibiting state.

Chip-based wide field-of-view total internal reflection fluorescence microscopy.

Conventional total internal reflection fluorescence (TIRF) microscopy requires either an oil-immersed objective with high numerical aperture or a bulky prism with high refractive index to generate the evanescent waves that work as the illumination source for fluorophores. Precise alignment of the optical path is necessary for optimizing the imaging performance of TIRF microscopy, which increases the operation complexity. In this Letter, a planar photonic chip composed of a dielectric multilayer and a scattering layer is proposed to replace the TIRF objective or the prism. The uniform evanescent waves can be excited under uncollimated incidence through this chip, which simplifies the alignment of the optical configurations and provides shadowless illumination. Due to the separation of the illumination and detection light paths, TIRF microscopy can have a large field-of-view (FOV).