Evanescent Field Illumination Research Articles

SNC‐TIRS: Label‐Free Single Nanoparticle Characterization System Based on Total Internal Reflection Scattering Signal

AbstractNanoparticles are used in various biomedical applications like in vitro diagnostics, imaging, drug delivery, and other therapeutic uses. The performance of such applications requires accurate knowledge of size distribution that needs precise characterization techniques. In this study, a single‐nanoparticle characterization system based on the total internal reflected scattering signal (SNC‐TIRS) is demonstrated for the size estimation of synthetic nanoparticles and exosomes. A compact waveguide‐based evanescent field illumination is used to convert a digital microscope into a nanoparticle characterization system based on scattering signals from diffusing nanoparticles in Brownian motion. By analyzing single‐particle trajectories, the system estimates the hydrodynamic size, average number of particles observed per unit volume, and the distribution of sample heterogeneity. SNC‐TIRS is benchmarked with inorganic nanoparticles and biomaterials in size range of 80–250 nm. Its utility to characterize the size of exosomes isolated from plasma using magnetic nanoparticles (MNPs) is shown. The hydrodynamic size of bare MNPs (123.83 ± 29.19 nm), antibody‐bound‐MNPs (161.33 ± 29.47 nm), and antibody‐exosome‐bound‐MNP conjugates (224.82 ± 25.89) are estimated and the results obtained agree with standard methods such as dynamic light scattering and nanoparticle tracking analysis. SNC‐TIRS can enable the label‐free characterization of nanomaterials and clinically relevant biomarkers.

Light scattering in TIRF microscopy: A theoretical study of the limits to surface selectivity

In TIRF microscopy, the sample resides near a surface in an evanescent optical field that, ideally, decreases in intensity with distance from the surface in a pure exponential fashion. In practice, multiple surfaces and imperfections in the optical system and refractive index (RI) inhomogeneities in the sample (often living cells) produce propagating scattered light that degrades the exponential purity. RI inhomogeneities cannot easily be avoided. How severe is the consequent optical degradation? Starting from Maxwell’s equations, we derive a first-order perturbative approximation of the electric field strength of light scattered by sample RI inhomogeneities of several types under coherent evanescent field illumination. The approximation provides an expression for the scattering field of any arbitrary RI inhomogeneity pattern. The scattering is not all propagating; some is evanescent and remains near the scattering centers. The results presented here are only a first-order approximation, and they ignore multiple scattering and reflections off the total internal reflection (TIR) surface. For simplicity, we assume that the RI variations in the z direction are insignificant within the depth of the evanescent field and consider only scattering of excitation light, not fluorescence emission light. The general conclusion of most significance from this study is that TIR scattering from a sample with RI variations typical of those on a cell culture alters the effective thickness of the illumination to only ∼50% greater than it would be without scattering. The qualitative surface selectivity of TIR fluorescence is largely retained even in the presence of scattering. Quantitatively, however, scattering will cause a deviation from the incident exponential decay at shorter distances, adding a slower decaying background. Calculations that assume a pure exponential decay will be approximations, and scattering should be taken into account. TIR scattering is only slightly dependent on polarization but is strongly reduced for the highest accessible incidence angles.

Lifetime Spectroscopy of Exosomes under Evanescent Field Illumination

Lifetime Spectroscopy of Exosomes under Evanescent Field Illumination

Orientational Imaging of a Single Gold Nanorod at the Liquid/Solid Interface with Polarized Evanescent Field Illumination.

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.

Surface Trafficking of APP and BACE in Live Cells

Amyloid‐β (Aβ)‐peptide, the major constituent of the plaques that develop during Alzheimer's disease, is generated via the cleavage of Aβ precursor protein (APP) by β‐site APP‐cleaving enzyme (BACE). Using live‐cell imaging of APP and BACE labeled with pH‐sensitive proteins, we could detect the release events of APP and BACE and their distinct kinetics. We provide kinetic evidence for the cleavage of APP by α‐secretase on the cellular surface after exocytosis. Furthermore, simultaneous dual‐color evanescent field illumination revealed that the two proteins are trafficked to the surface in separate compartments. Perturbing the membrane lipid composition resulted in a reduced frequency of exocytosis and affected BACE more strongly than APP. We propose that surface fusion frequency is a key factor regulating the aggregation of APP and BACE in the same membrane compartment and that this process can be modulated via pharmacological intervention.

Kv2.1 Cell Surface Clusters Promote Maturation of Clathrin-Coated Pits

The voltage-gated potassium channel Kv2.1 localizes to stable, micro-domains on the cell surface where it plays a non-conducting role. These surface structures are specialized platforms involved in trafficking of Kv channels to and from the cell surface in hippocampal neurons and transfected HEK cells [Deutsch et al., MBoC 15, pp 2917-29 (2012)]. Internalization of Kv2.1 occurs through clathrin-mediated endocytosis and clathrin-coated pits (CCP) localizes adjacent to these micro-domains. This study examines the relationship between Kv2.1 clusters and CCP maturation.TIRF-microscopy was used to study GFP-tagged-clathrin light chain CCPs in live HEK293 cells. HEK cells do not express endogenous Kv2.1, making them a suitable model system in which to investigate the role of Kv2.1 in clathrin-mediated endocytosis. We tracked individual CCPs and measured their lifetimes. This analysis is obtained from the appearance and disappearance of GFP fluorescence within the evanescent field illumination. We compare the dynamics of CCPs in control cells transfected with GFP-CLC and cells co-transfected with Kv2.1 or a Kv2.1 mutant lacking the last 318 amino acids of the C-terminus (ΔC-Kv2.1) necessary for cluster formation.In control cells, CCPs had a mean lifetime of 12.6±0.3 s (mean±sem). The lifetime of CCPs in cells co-expressing Kv2.1 and GFP-CLC was reduced by 50%. When GFP-CLC was co-transfected with the non-clustering ΔC-Kv2.1, the lifetime of CCPs increased by 17%. ΔC-Kv2.1 also decreased the rate of channel endocytosis by 12%.These data reveal that Kv2.1, specifically the C-terminal tail, has a direct effect on CCP lifetimes and thereby maturation. Cells expressing clustering Kv2.1 exhibit more rapidly maturing CCPs as seen by the rate of Kv2.1 internalization. Non-clustering Kv2.1 increases CCP lifetimes, and complete loss of Kv2.1 results in even longer lifetimes, i.e. slower maturation. These results indicate that Kv2.1 cluster formation remodels clathrin-mediated endocytosis.

Slab waveguide photobioreactors for microalgae based biofuel production

Microalgae are a promising feedstock for sustainable biofuel production. At present, however, there are a number of challenges that limit the economic viability of the process. Two of the major challenges are the non-uniform distribution of light in photobioreactors and the inefficiencies associated with traditional biomass processing. To address the latter limitation, a number of studies have demonstrated organisms that directly secrete fuels without requiring organism harvesting. In this paper, we demonstrate a novel optofluidic photobioreactor that can help address the light distribution challenge while being compatible with these chemical secreting organisms. Our approach is based on light delivery to surface bound photosynthetic organisms through the evanescent field of an optically excited slab waveguide. In addition to characterizing organism growth-rates in the system, we also show here, for the first time, that the photon usage efficiency of evanescent field illumination is comparable to the direct illumination used in traditional photobioreactors. We also show that the stackable nature of the slab waveguide approach could yield a 12-fold improvement in the volumetric productivity.

Fluorescent vesicles for signal amplification in reverse phase protein microarray assays

Developments in microarray technology promise to lead to great advancements in the biomedical and biological field. However, implementation of these analytical tools often relies on signal amplification strategies that are essential to reach the sensitivity levels required for a variety of biological applications. This is true especially for reverse phase arrays where a complex biological sample is directly immobilized on the chip. We present a simple and generic method for signal amplification based on the use of antibody-tagged fluorescent vesicles as labels for signal generation. To assess the gain in assay sensitivity, we performed a model assay for the detection of rabbit immunoglobulin G (IgG) and compared the limit of detection (LOD) of the vesicle assay with the LOD of a conventional assay performed with fluorescent reporter molecules. We evaluated the improvements for two fluorescence-based transduction setups: a high-sensitivity microarray reader (ZeptoREADER) and a conventional confocal scanner. In all cases, our strategy led to an increase in sensitivity. However, gain in sensitivity widely depended on the type of illumination; whereas an approximately 2-fold increase in sensitivity was observed for readout based on evanescent field illumination, the contribution was as high as more than 200-fold for confocal scanning.

A versatile liquid-core/liquid-twin-cladding waveguide micro flow cell fabricated by rapid prototyping

In this paper we present the design and operation of a micro flow cell comprising a liquid-core/liquid-twin-cladding waveguide for on-chip fluorescence spectroscopy based on evanescent field illumination. The application of an inner (sample) and outer cladding stream minimizes the sample volume for optical measurements and ensures the analyte position in the evanescent field for excitation at the core/cladding interface. The fiber-chip-coupled laser light is guided by the fluidic waveguide providing a uniform excitation along the analysis channel. Fluorescence intensity measurements of different sample solutions were conducted to illustrate the operational quality. The fluidics device is fabricated by laser microstereolithography in 1.5 h.

Measurement of nanoparticle sizes by conventional optical microscopy with standing evanescent field illumination

The size of a particle smaller than the diffraction limit is measured using a conventional optical microscope by adopting a standing evanescent field illumination. The scattering intensity from a nanoparticle is periodically modulated by shifting the intensity fringes of the standing evanescent field. By measuring contrast of scattering intensity variation during one cycle of modulation, particle sizes can be estimated easily. Furthermore, material dependence can be canceled using contrast as an evaluation factor. From the experimental results, particle sizes ranging from 20 to 250 nm are successfully determined. This technique offers a low-cost size measurement for nanoparticles.

Coupling between Clathrin-Coated-Pit Invagination, Cortactin Recruitment, and Membrane Scission Observed in Live Cells

During clathrin-mediated endocytosis, membrane scission marks the isolation of a cargo-laden clathrin-coated pit (CCP) from the cell exterior. Here we used live-cell imaging of a pH-sensitive cargo to visualize the formation of clathrin-coated vesicles (CCVs) at single CCPs with a time resolution of seconds. We show that CCPs are highly dynamic and can produce multiple vesicles in succession. Using alternating evanescent field and epifluorescence illumination, we show that CCP invagination and scission are tightly coupled, with scission coinciding with maximal displacement of CCPs from the plasma membrane and with peak recruitment of cortactin-DsRed, a dynamin and F-actin binding protein. Finally, perturbing actin polymerization with latrunculin-B drastically reduces the efficiency of membrane scission and affects many aspects of CCP dynamics. We propose that CCP invagination, actin polymerization, and CCV formation are highly coordinated for efficient endocytosis.

Water Secretion Associated with Exocytosis in Endocrine Cells Revealed by Micro Forcemetry and Evanescent Wave Microscopy

It has been a long belief that release of substances from the cell to the extracellular milieu by exocytosis is completed by diffusion of the substances from secretory vesicles through the fusion pore. Involvement of any mechanical force that may be superposed on the diffusion to enhance the releasing process has not been elucidated to date. We tackled this problem in cultured bovine chromaffin cells using direct and sensitive methods: the laser-trap forcemetry and the evanescent-wave fluorescence microscopy. With a laser beam, we trapped a micro bead in the vicinity of a cell (with 1μm of separation) and observed movements of the bead optically. Electrical stimulation of the cell induced many of rapid and transient movements of the bead in a direction away from the cell surface. Upon the same stimulation, secretory vesicles stained with a fluorescent probe, acridine orange, and excited under the evanescent field illumination, showed a flash-like response: a transient increase in fluorescence intensity associated with a diffuse cloud of brightness, followed by a complete disappearance. These mechanical and fluorescence transients indicate a directional flow of substances. Blockers of the Cl− channel suppressed the rates of both responses in a characteristic way but not exocytotic fusion itself. Immunocytochemical studies revealed the presence of Cl− and K+ channels on the vesicle membranes. These results suggest that the externalization of hormones or transmitters upon exocytosis of vesicles is augmented by secretion of water from the vesicle membrane through the widened fusion pore, possibly modulating the rate and reach of the hormone or transmitter release and facilitating transport of the signal molecules in intercellular spaces.

Electromagnetic force on a metallic particle in the presence of a dielectric surface

By using a method, previously established to calculate electromagnetic fields, we compute the force of light upon a metallic particle. This procedure is based on both Maxwell's Stress Tensor and the Couple Dipole Method. With these tools, we study the force when the particle is over a flat dielectric surface. The multiple interaction of light between the particle and the surface is fully taken into account. The wave illuminating the particle is either evanescent or propagating depending an whether or not total internal reflection takes place. We analyze the behaviour of this force on either a small or a large particle in terms of the wavelength. A remarkable result obtained for evanescent field illumination, is that the force on a small silver particle can be either attractive or repulsive depending on the wavelength. This behaviour also varies as the particle becomes larger.

Colloidal Particles at Water-Glass Interface: Deposition Kinetics and Surface Heterogeneity

Videomicroscopy in combination with evanescent field illumination is applied to study the sorption of colloidal particles from flow in a parallel plate channel on a glass surface. The experiments, carried out in the presence of a repulsive electrostatic barrier, reveal surprisingly complex results: The glass surface, though optically flat and well cleaned, is not homogeneous, but rather the sorption occurs at a limited number of preferred sites. Moreover, these sites are not static: new sites keep appearing at random positions on the observed surface and disappearing at a rate ofkd= 1.3 × 10−5s−1. These findings can be understood within a simple model that takes into account slow but inevitable dissolution of the glass surface. The bulk glass contains potential adsorbers, which are continuously being exposed by the dissolution process and act as transient adsorption sites, before being washed off by the flowing buffer solution.

Enhanced sensitivity near-field scanning optical microscopy at high spatial resolution

An apertureless near-field optical-imaging method is presented that achieves high spatial resolution as well as a ∼4000-fold increase in detection sensitivity, by exploiting the highly localized enhanced near-field interactions between the sample (e.g., Au nanospheres) and a sharp atomic force microscope tip under evanescent laser field illumination. This represents a general method for optical imaging at ⩽2 nm spatial resolution, and is applicable to both resonant (i.e., scattering) as well as nonresonant (i.e., fluorescence, Raman, etc.) spectroscopic methods.

Holographic pattern photobleaching apparatus for the measurement of lateral transport processes at interfaces—Design and performance

A new instrument was developed which allows the detection and characterization of lateral transport processes at the fluid–solid interface. It is based on holographic photobleaching combined with a laterally resolved fluorescence detection by a photon-counting camera. The geometry of the holographic pattern was chosen such that by ‘‘on-chip’’ integration of photoelectrons in the liquid-nitrogen-cooled camera the images are integrated along the symmetry axis of the pattern and subsequently stored on the chip. Time-dependent changes in the pattern caused by directed motion or by diffusion are then qualified by Fourier analysis. Lateral diffusion coefficients between 10−12 and 10−6 cm2/s and velocities between 10−10 and 10−4 m/s are measurable. In order to discriminate processes at the surface and in the volume, evanescent field illumination can be chosen. The penetration depth of the evanescent field can be varied from 96 to 800 nm. The performance of the instrument is demonstrated on selected examples.