Sub-diffraction Limit Research Articles

Scalable Method for the Fabrication and Testing of Glass-Filled, Three-Dimensionally Sculpted Extraordinary Transmission Apertures

This Letter features a new, scalable fabrication method and experimental characterization of glass-filled apertures exhibiting extraordinary transmission. These apertures are fabricated with sizes, aspect ratios, shapes, and side-wall profiles previously impossible to create. The fabrication method presented utilizes top-down lithography to etch silicon nanostructures. These nanostructures are oxidized to provide a transparent template for the deposition of a plasmonic metal. Gold is deposited around these structures, reflowed, and the surface is planarized. Finally, a window is etched through the substrate to provide optical access. Among the structures created and tested are apertures with height to diameter aspect ratios of 8:1, constructed with rectangular, square, cruciform, and coupled cross sections, with tunable polarization sensitivity and displaying unique properties based on their sculpted side-wall shape. Transmission data from these aperture arrays is collected and compared to examine the role of spacing, size, and shape on their overall spectral response. The structures this Letter describes can have a variety of novel applications from the creation of new types of light sources to massively multiplexed biosensors to subdiffraction limit imaging techniques.

Sub‐Diffraction Limit Imaging of Inorganic Nanowire Networks Interfacing Cells

Sub-diffraction limit imaging of SnO2 nanowires (NWs) interfacing cells is studied using stochastic optical reconstruction microscopy. This method allows the estimation of the dimension of the NWs with a rather high resolution compared with previous imaging methods. In addition, since this strategy is compatible with biological environments, the NWs can be imaged with cell membrane proteins simultaneously and the effect of the NWs on the cell adhesion efficiency can be analyzed.

A Super Lens System for Demagnification Imaging Beyond the Diffraction Limit

A super lens system is proposed to achieve subdiffraction limit demagnification imaging. The super lens system consists of a hyperlens with planar input and output surfaces, a metal superlens, and a plasmonic reflector. By employing the hyperlens to transform evanescent waves into propagating waves and employing the metal superlens and the plasmonic reflector to amplify evanescent waves, the super lens system can produce a subdiffraction limit image with relatively high electric field intensity. The reduction factor of the super lens system depends on the geometric parameters of the hyperlens. Simulation results show that an image with a half-pitch resolution of about one tenth the operating wavelength and a reduction factor of about 2.2 can be produced by the super lens system. The proposed super lens system has potential applications in nanolithography.

Mechanisms of plasmon-enhanced femtosecond laser nanoablation of silicon

We perform plasmon-enhanced femtosecond laser ablation of silicon using gold nanorods to produce sub-diffraction limit features. While the observed hole shape seems inconsistent with calculated field distribution, we show that using a carrier diffusion-based model, both shape and depth of the nanoholes can be reliably explained. The laser energy is first deposited into electron-hole pairs that are created in the nanostructure's enhanced near-field. Those carriers then diffuse and transfer their energy to the silicon lattice, producing ablation. Increased importance of the carrier diffusion process is shown to arise from the extreme localization of the deposited energy around the nanostructure, due to the plasmonic effect. The characteristic shape of holes is revealed as a striking signature of the screened charge carriers-phonon coupling that is shown to channel the heat transfer to the lattice and control ablation.

Single-image far-field subdiffraction limit imaging with axicon

This Letter presents a technique for subdiffraction limit imaging termed Bessel beam microscopy (BBM). By placing a lens in series with an axicon in the optical path of a microscope, the diffraction-limited resolution of the base microscope is improved by one third. This improvement is demonstrated experimentally by resolving individual subdiffraction limit fluorescent beads in a close-pack arrangement. The behavior of the BBM system is explored using angular diffraction simulations, demonstrating the possibility of resolving features spaced as little as 110 nm apart when viewed with a 100×1.4 NA objective. Unique among super-resolution techniques, BBM acquires subdiffraction limit information in a single image with broadband unstructured illumination using only static geometric optics placed between the microscope and camera.

Tuning the 3D plasmon field of nanohole arrays

Modern photonics is being revolutionized through the use of nanostructured plasmonic materials, which confine light to sub-diffraction limit resolution providing universal, sensitive, and simple transducers for molecular sensors. Understanding the mechanisms by which light interacts with plasmonic crystals is essential for developing application-focussed devices. The strong influence of grating coupling on electromagnetic field distribution, frequency and degeneracy of plasmon bands has now been characterized using hexagonal nanohole arrays. An equation for nanohole arrays was derived to demonstrate the strong influence of incidence and rotation angle on optical properties of 2D plasmonic crystals such as nanohole arrays. Consequently, we report experimental data that are in strong agreement with finite difference time-domain (FDTD) simulations that clearly demonstrate the influence of the grating coupling conditions on the optical properties (such as plasmon degeneracy and bandwidth), and on the distribution of the plasmon field around nanohole arrays (including tuneable penetration depths and highly localized fields). The tuneable 3D plasmon field allowed for controlled sensing properties and by increasing the angle of incidence to 30 degrees, the resonance wavelength was tuned from 1000 to 600 nm, and the sensitivity was enhanced by nearly 300% for a protein assay using surface plasmon resonance (SPR) and by 40% with surface-enhanced Raman scattering (SERS) sensors.

Super-resolution Optical Measurement of Nanoscale Photoacid Distribution in Lithographic Materials

We demonstrate a method using photoactivation localization microscopy (PALM) in a soft-material system, with a rhodamine-lactam dye that is activated by both ultraviolet light and protonation, to reveal the nanoscale photoacid distribution in a model photoresist. Chemically amplified resists are the principal lithographic materials used in the semiconductor industry. The photoacid distribution generated upon exposure and its subsequent evolution during post-exposure bake is a major limiting factor in determining the resolution and lithographic quality of the final developed resist image. Our PALM data sets resolve the acid distribution in a latent image with subdiffraction limit accuracy. Our overall accuracy is currently limited by residual mechanical drift.

Sub-diffraction Limit Localization of Proteins in Volumetric Space Using Bayesian Restoration of Fluorescence Images from Ultrathin Specimens

Photon diffraction limits the resolution of conventional light microscopy at the lateral focal plane to 0.61λ/NA (λ = wavelength of light, NA = numerical aperture of the objective) and at the axial plane to 1.4nλ/NA2 (n = refractive index of the imaging medium, 1.51 for oil immersion), which with visible wavelengths and a 1.4NA oil immersion objective is ∼220 nm and ∼600 nm in the lateral plane and axial plane respectively. This volumetric resolution is too large for the proper localization of protein clustering in subcellular structures. Here we combine the newly developed proteomic imaging technique, Array Tomography (AT), with its native 50–100 nm axial resolution achieved by physical sectioning of resin embedded tissue, and a 2D maximum likelihood deconvolution method, based on Bayes' rule, which significantly improves the resolution of protein puncta in the lateral plane to allow accurate and fast computational segmentation and analysis of labeled proteins. The physical sectioning of AT allows tissue specimens to be imaged at the physical optimum of modern high NA plan-apochormatic objectives. This translates to images that have little out of focus light, minimal aberrations and wave-front distortions. Thus, AT is able to provide images with truly invariant point spread functions (PSF), a property critical for accurate deconvolution. We show that AT with deconvolution increases the volumetric analytical fidelity of protein localization by significantly improving the modulation of high spatial frequencies up to and potentially beyond the spatial frequency cut-off of the objective. Moreover, we are able to achieve this improvement with no noticeable introduction of noise or artifacts and arrive at object segmentation and localization accuracies on par with image volumes captured using commercial implementations of super-resolution microscopes.

Dual-Stage Lithography from a Light-Driven, Plasmon-Assisted Process: A Hierarchical Approach to Subwavelength Features

A hierarchy of lithographic-type imaging generating 3 μm lines incorporating subdiffraction limit features was obtained through a novel two-step reaction process. Photochemically generated ketyl radicals were used to make defined lines of silver nanoparticles. The excitation of nanoparticle surface plasmons was then used to generate highly localized heat that causes polymerization selectively on the surfaces of excited particles. The nylon-6 polymer that is generated serves as a solubility switch used to retain the features on the substrate selectively; various imaging techniques were used to establish the nature of the nylon shells. This work shows that the heat generated by plasmon excitation can be exploited to generate negative-type lithographic features with dimensions well below the diffraction limit.

Frequency Shifts in SERS for Biosensing

We report an observation of a peculiar effect in which the vibrational frequencies of antibody-conjugated SERS-active reporter molecules are shifted in quantitative correlation with the concentration of the targeted antigen. We attribute the frequency shifts to mechanical perturbations in the antibody-reporter complex, as a result of antibody-antigen interaction forces. Our observation thus demonstrates the potentiality of an antibody-conjugated SERS-active reporter complex as a SERS-active nanomechanical sensor for biodetection. Remarkably, our sensing scheme, despite employing only one antibody, was found to be able to achieve detection sensitivity comparable to that of a conventional sandwich immunoassay. Additionally, we have carried out a proof-of-concept study into using multiple "stress-sensitive" SERS reporters for multiplexed detection of antigen-antibody bindings at the subdiffraction limit. The current work could therefore pave the way to realizing a label-free high-density protein nanoarray.

Plasmonic-Enhanced Molecular Fluorescence within Isolated Bowtie Nano-Apertures

We report experimental behaviors of polarization-dependent, plasmonic-enhanced molecular fluorescence within isolated bowtie nano-apertures (BNAs) milled in aluminum films. BNAs provide efficient control of the fluorescent count rate per molecule and the decay lifetime of the molecules and provide an effective detection volume at the nanometer scale by tuning either the excitation light polarization or the BNA size. Interestingly, large BNAs (>300 nm) present high plasmonic-enhanced fluorescence efficiency and can simultaneously confine the detection volume below the subdiffraction limit. Numerical simulations were performed that agreed qualitatively with the experimental results. The BNAs have potential applications, especially for single-molecule biological analysis.

Identification of the factors affecting co-localization precision for quantitative multicolor localization microscopy

This work explores the potential of multi-color Photoactivated Localization Microscopy (PALM) imaging to probe sub-diffraction limit interactions between proteins with spectrally separated labels. Using a PALM setup built around a commercial microscope axially stabilized to nm-level, we determined the ultimate registration accuracy that could be achieved (10 nm) and compared the performance of three different pairs of fluorescent proteins that can be used in dual color PALM. Fusion constructs were cloned and imaged either in vitro or at the cell plasma membrane, allowing to identify a current limit to co-localization precision of approximately 30–40 nm. We identified the better performing pair and present a concluding perspective application to a co-clustering study.

A polychromatic approach to far-field superlensing at visible wavelengths

Breaking the diffraction barrier in the visible part of the electromagnetic spectrum is of fundamental importance. Far-field subwavelength focusing of light could, for instance, drastically broaden the possibilities available in nanolithography, light-matter interactions and sensing at the nanoscale. Similarly, imaging with a nanometric resolution could result in incredible breakthroughs in soft matter and biology. There have been numerous proposals in this regard based on metamaterials, structured illumination methods or diffractive optical components. The common denominator of all these approaches resides in their monochromatic nature. Here we show that using polychromatic light in dispersive metamaterials allows us to circumvent many limitations associated with previous monochromatic approaches. We design a plasmonic metalens based on metallic nanorods that, when used with broadband light fields, can beat the diffraction limit for imaging and focusing from the far field.

Optimal 3D single-molecule localization for superresolution microscopy with aberrations and engineered point spread functions

Photo-activation localization microscopy is a far-field superresolution imaging technique based on the localization of single molecules with subdiffraction limit precision. Known under acronyms such as PALM (photo-activated localization microscopy) or STORM (stochastic optical reconstruction microscopy), these techniques achieve superresolution by allowing only a sparse, random set of molecules to emit light at any given time and subsequently localizing each molecule with great precision. Recently, such techniques have been extended to three dimensions, opening up unprecedented possibilities to explore the structure and function of cells. Interestingly, proper engineering of the three-dimensional (3D) point spread function (PSF) through additional optics has been demonstrated to theoretically improve 3D position estimation and ultimately resolution. In this paper, an optimal 3D single-molecule localization estimator is presented in a general framework for noisy, aberrated and/or engineered PSF imaging. To find the position of each molecule, a phase-retrieval enabled maximum-likelihood estimator is implemented. This estimator is shown to be efficient, meaning it reaches the fundamental Cramer-Rao lower bound of x, y, and z localization precision. Experimental application of the phase-retrieval enabled maximum-likelihood estimator using a particular engineered PSF microscope demonstrates unmatched low-photon-count 3D wide-field single-molecule localization performance.

Differential Impact of Caveolae and Caveolin-1 Scaffolds on The Membrane Raft Proteome

Caveolae, a class of cholesterol-rich lipid rafts, are smooth invaginations of the plasma membrane whose formation in nonmuscle cells requires caveolin-1 (Cav1). The recent demonstration that Cav1-associated cavin proteins, in particular PTRF/cavin-1, are also required for caveolae formation supports a functional role for Cav1 independently of caveolae. In tumor cells deficient for Golgi β-1,6N-acetylglucosaminyltransferase V (Mgat5), reduced Cav1 expression is associated not with caveolae but with oligomerized Cav1 domains, or scaffolds, that functionally regulate receptor signaling and raft-dependent endocytosis. Using subdiffraction-limit microscopy, we show that Cav1 scaffolds are homogenous subdiffraction-limit sized structures whose size distribution differs from that of Cav1 in caveolae expressing cells. These cell lines displaying differing Cav1/caveolae phenotypes are effective tools for probing the structure and composition of caveolae. Using stable isotope labeling by amino acids in cell culture, we are able to quantitatively distinguish the composition of caveolae from the background of detergent-resistant membrane proteins and show that the presence of caveolae enriches the protein composition of detergent-resistant membrane, including the recruitment of multiple heterotrimeric G-protein subunits. These data were further supported by analysis of immuno-isolated Cav1 domains and of methyl-β-cyclodextrin-disrupted detergent-resistant membrane. Our data show that loss of caveolae results in a dramatic change to the membrane raft proteome and that this change is independent of Cav1 expression. The proteomics data, in combination with subdiffraction-limit microscopy, indicates that noncaveolar Cav1 domains, or scaffolds are structurally and functionally distinct from caveolae and differentially impact on the molecular composition of lipid rafts.

Optimal measurement constellation of the fluoroBancroft localization algorithm for position estimation in tracking confocal microscopy

The fluoroBancroft algorithm is an analytical approach that converts a collection of fluorescence intensity measurements generated by an isolated sub-diffraction limit source into an estimate with nanometer-scale precision of the source position. Based on this algorithm, we have developed a scheme for tracking single fluorescent particles in a confocal microscope. In this paper, we determine an optimal measurement constellation for the estimation algorithm. The position estimation bias and uncertainty arising from the photon counting statistics are calculated based on the assumption that the natural logarithm of a Poisson random variable with large rate can be approximated as a random variable with a Gaussian distribution. A sufficient condition for an unbiased measurement constellation and the optimal radius of a given constellation geometry with six measurements are then derived. The results are illustrated through both numerical simulation and experiments.

Optical cavity characterization in nanowires via self-generated broad-band emission

Broadband white light is of great spectroscopic value and would be a powerful tool for nanoscale spectroscopy, however, generation and direction of white light on this length scale remains challenging. Here, we demonstrate the generation of broadband white light in sub-wavelength diameter Gallium Nitride (GaN) wires by coincident one- and two-photon absorption mediated via defect states. This generation of broadband, "white" light enables single-nanowire interferometric measurements of the nanowires themselves via analysis of the Fabry-Pérot fringes that overlay the entirety of the emission spectrum. The quality factor and finesse of individual nanowire cavities were measured and calculated to be 186 ± 88 and 3.05 ±0.6 respectively, averaged over 20 individual wires. This work presents a new, simple approach for the generation and direction of broad band white light at sub-diffraction limit length scales, ideal for translating classical white light spectroscopies to higher spatial resolutions then previously achieved.

High Speed Resolution Enhancement by Optical Pupil Function Modulating System

Superresolution fluorescent microscopy (nanoscopy) offers unprecedented resolution of biological structures by overcoming the fundamental diffraction limit of light. There are several superresolution technologies like stimulated emission depletion (STED), structured illumination microscopy (SIM), stochastic optical reconstruction microscopy (STORM) and fluorescence photoactivation localization (FPALM) all show promise as they offer subdiffraction limit optical resolution. The further challenge in superresolution is the improvements of the temporal resolution, because the temporal resolutions of the above four methods are too low to pursue the movements of proteins at ms timescales. However, it looks so difficult because FPALM and STORM are based on single molecular detection and stochastic photoswitching while STED and SIM require laser illumination scanning. Here, we constructed a microscopic system that can voluntarily modulate optical transmission of the pupil function by placing a reflective liquid crystal micro mirror array (LCMM) onto the Fourier plane that alters the point spread function (PSF) shape. It has been reported that the diffraction limit was overcome by subtracting the normal PSF from the low-pass filtered PSF (∗). Our present system can obtain a subtracted image of superresolve-masked image and the low-pass filtered image within one frame of a camera. We achieved 2 ms temporal resolution at 120 nm spatial resolution without any special probes and scanning systems. Furthermore, because the present technique is compatible with fluorescent probes that lack polarization or coherency, we can apply it to various biological investigations. In this meeting, we show basic principles and constructions of our present nanoscopy, and demonstrate the potentials of it for live cell imaging both on the membrane and intracellular, and for single particle tracking.(∗) Heintzmann R, Sarafis V, Munroe P, Nailon J, Hanley QS, Jovin TM., Micron. 34,293-300 (2003).

Point Spread Diffraction Patterns and Super-Resolution Particle Localization

Sub-diffraction limit localization of molecules within cells depends upon fitting the observed photon distribution to the point spread function. As such, accurate knowledge of the point spread function is important to super-resolution microscopy and critical to determining the trajectories and dynamics of molecules within cells. While simple geometrical optics serves well to represent light propagation on the macroscopic level, more elaborate wave representations are necessary to describe light propagation within a few wavelengths of focal points, such as single fluorophores imaged by microscope objectives. As a result, numerous theoretical approximations to experimental point spread functions exist as the exact theoretical point spread function is unknown. Further consideration must be given that for realistic in vivo experiments, frequently events of interest will not entirely match the design conditions of the microscope; most events of interest will not be perfectly in focus, nor will the index of refraction within the cell necessarily match that of the medium. Additionally, a number of imaging modes explicitly rely upon out-of-focus images for three dimensional localization. Here, through serial sectioning microscopy, we explore the experimental point spread function in comparison with various models, determining which models are robust and provide accurate sub-diffraction limit localization for realistic data.

Light-trapping nano-structures in organic photovoltaic cells

We review nano-structures used as light trapping schemes in organic photovoltaic (OPV) cells. Light management via nano-structures enables one to exploit sub-diffraction limit properties, thereby giving access to enhanced light absorption in OPV cells. Light trapping schemes have been demonstrated in the form of surface plasmons, anti-reflection coatings, and photonic crystal patterns. Versatile methods are now available to facilitate fabrication of such nano-structures on sufficiently large areas for OPV applications.