Diffraction Limit Of Light Research Articles

Super-Resolution-Chip: an in-vitro platform that enables super-resolution microscopy of co-cultures and 3D systems.

The development of organs-on-a-chip platforms has revolutionized in-vitro cellular culture by allowing cells to be grown in an environment that better mimics human physiology. However, there is still a challenge in integrating those platforms with advanced imaging technology. This is extremely important when we want to study molecular changes and subcellular processes on the level of a single molecule using super-resolution microscopy (SRM), which has a resolution beyond the diffraction limit of light. Currently, existing platforms that include SRM have certain limitations, either as they only support 2D monocultures, without flow or as they demand a lot of production and handling. In this study, we developed a Super-Res-Chip platform, consisting of a 3D-printed chip and a porous membrane, that could be used to co-culture cells in close proximity either in 2D or in 3D while allowing SRM on both sides of the membrane. To demonstrate the functionality of the device, we co-cultured in endothelial and epithelial cells and used direct stochastic optical reconstruction microscopy (dSTORM) to investigate how glioblastoma cells affect the expression of the gap-junction protein Connexin43 in endothelial cells grown in 2D and in 3D. Cluster analysis of Connexin43 distribution revealed no difference in the number of clusters, their size, or radii, but did identify differences in their density. Furthermore, the spatial resolution was high also when the cells were imaged through the membrane (20-30 nm for x-y) and 10-20 nm when imaged directly both for 2D and 3D conditions. Overall, this chip allows to characterize of complex cellular processes on a molecular scale in an easy manner and improved the capacity for imaging in a single molecule resolution complex cellular organization.

Terahertz phononic crystal in plasmonic nanocavity

Interaction between photons and phonons in cavity optomechanical systems provides a new toolbox for quantum information technologies. A GaAs/AlAs pillar multi-optical mode microcavity optomechanical structure can obtain phonons with ultra-high frequency (~THz). However, the optical field cannot be effectively restricted when the diameter of the GaAs/AlAs pillar microcavity decreases below the diffraction limit of light. Here, we design a system that combines Ag nanocavity with GaAs/AlAs phononic superlattices, where phonons with the frequency of 4.2 THz can be confined in a pillar with ~4 nm diameter. The Q c/V reaches 0.22 nm−3, which is ~80 times that of the photonic crystal (PhC) nanobeam and ~100 times that of the hybrid point-defect PhC bowtie plasmonic nanocavity, where Q c is optical quality factor and V is mode volume. The optomechanical single-photon coupling strength can reach 12 MHz, which is an order of magnitude larger than that of the PhC nanobeam. In addition, the mechanical zero-point fluctuation amplitude is 85 fm and the efficient mass is 0.27 zg, which is much smaller than the PhC nanobeam. The phononic superlattice-Ag nanocavity optomechanical devices hold great potential for applications in the field of integrated quantum optomechanics, quantum information, and terahertz-light transducer.

Expandable ELAST for super-resolution imaging of thick tissue slices using a hydrogel containing charged monomers

Hydrogels have been utilized extensively as a material for retaining position information in tissue imaging procedures, such as tissue clearing and super-resolution imaging. Immunostaining thick biological tissues, however, poses a bottleneck that restricts sample size. The recently developed technique known as entangled link-augmented stretchable tissue-hydrogel (ELAST) accelerates the immunostaining process by embedding specimens in long-chain polymers and stretching them. A more advanced version of ELAST, magnifiable entangled link-augmented stretchable tissue-hydrogel (mELAST), achieves rapid immunostaining and tissue expansion by embedding specimens in long-chain neutral polymers and subsequently hydrolyzing them. Building on these techniques, we introduce a variant of mELAST called ExELAST. This approach uses charged monomers to stretch and expand tissue slices. Using ExELAST, we first tested two hydrogel compositions that could permit uniform expansion of biological specimens. Then, we apply the tailored hydrogel to the 500-μm-thick mouse brain slices and demonstrated that they can be stained within two days and imaged with a resolution below the diffraction limit of light.

Quantitative super-resolution microscopy reveals the differences in the nanoscale distribution of nuclear phosphatidylinositol 4,5-bisphosphate in human healthy skin and skin warts.

Introduction: Imaging of human clinical formalin-fixed paraffin-embedded (FFPE) tissue sections provides insights into healthy and diseased states and therefore represents a valuable resource for basic research, as well as for diagnostic and clinical purposes. However, conventional light microscopy does not allow to observe the molecular details of tissue and cell architecture due to the diffraction limit of light. Super-resolution microscopy overcomes this limitation and provides access to the nanoscale details of tissue and cell organization. Methods: Here, we used quantitative multicolor stimulated emission depletion (STED) nanoscopy to study the nanoscale distribution of the nuclear phosphatidylinositol 4,5-bisphosphate (nPI(4,5)P2) with respect to the nuclear speckles (NS) marker SON. Results: Increased nPI(4,5)P2 signals were previously linked to human papillomavirus (HPV)-mediated carcinogenesis, while NS-associated PI(4,5)P2 represents the largest pool of nPI(4,5)P2 visualized by staining and microscopy. The implementation of multicolor STED nanoscopy in human clinical FFPE skin and wart sections allowed us to provide here the quantitative evidence for higher levels of NS-associated PI(4,5)P2 in HPV-induced warts compared to control skin. Discussion: These data expand the previous reports of HPV-induced increase of nPI(4,5)P2 levels and reveal for the first time the functional, tissue-specific localization of nPI(4,5)P2 within NS in clinically relevant samples. Moreover, our approach is widely applicable to other human clinical FFPE tissues as an informative addition to the classical histochemistry.

Advanced Microscopy Techniques for Molecular Biophysics.

Though microscopy is most often intended as a technique for providing qualitative assessment of cellular and subcellular properties, when coupled with other instruments such as wavelength selectors, lasers, photoelectric devices and computers, it can perform a wide variety of quantitative measurements, which are demanding in establishing relationships between the properties and structures of biological material in all their spatial and temporal complexities. These combinations of instruments are a powerful approach to improve non-destructive investigations of cellular and subcellular properties (both physical and chemical) at a macromolecular scale resolution. Since many subcellular compartments in living cells are characterized by structurally organized molecules, this review deals with three advanced microscopy techniques well-suited for these kind of investigations, i.e., microspectrophotometry (MSP), super-resolution localization microscopy (SRLM) and holotomographic microscopy (HTM). These techniques can achieve an insight view into the role intracellular molecular organizations such as photoreceptive and photosynthetic structures and lipid bodies play in many cellular processes as well as their biophysical properties. Microspectrophotometry uses a set-up based on the combination of a wide-field microscope and a polychromator, which allows the measurement of spectroscopic features such as absorption spectra. Super resolution localization microscopy combines dedicated optics and sophisticated software algorithms to overcome the diffraction limit of light and allow the visualization of subcellular structures and dynamics in greater detail with respect to conventional optical microscopy. Holotomographic microscopy combines holography and tomography techniques into a single microscopy set-up, and allows 3D reconstruction by means of the phase separation of biomolecule condensates. This review is organized in sections, which for each technique describe some general aspects, a peculiar theoretical aspect, a specific experimental configuration and examples of applications (fish and algae photoreceptors, single labeled proteins and endocellular aggregates of lipids).

Optical Reading of Nanoscale Magnetic Bits in an Integrated Photonic Platform

In this paper, we propose a compact integrated hybrid plasmonic-photonic device for optical reading of nanoscale magnetic bits with perpendicular magnetic anisotropy in a magnetic racetrack on top of a photonic waveguide on the indium phosphide membrane on silicon platform. The hybrid device is constructed by coupling a doublet of V-shaped gold plasmonic nanoantennas on top of the indium phosphide waveguide. By taking advantage of the localized surface plasmons, our hybrid device can enable detection of the magnetization state in magnetic bits beyond the diffraction limit of light and enhance the polar magneto-optical Kerr effect (PMOKE). We further illustrate how combining the hybrid device with a plasmonic polarization rotator provides magneto-optical read-out by transforming the PMOKE-induced polarization change into an intensity variation of the waveguide mode. According to the simulation results based on a three-dimensional finite-difference time-domain method, the hybrid device can detect the magnetization states in targeted bits in a magnetic racetrack medium down to ~ 100x100 nm2, regardless of the magnetization state of the rest of the racetrack with a relative intensity contrast of greater than 0.5% for a ~ 200x100 nm2 magnetic bit. We believe our hybrid device can be an enabling technology that can connect integrated photonics with nanoscale spintronics, paving the way toward ultrafast and energy efficient advanced on-chip applications.

Vertical Emitting Nanowire Vector Beam Lasers.

Due to the peculiar structured light field with spatially variant polarizations on the same wavefront, vector beams (VBs) have sparked research enthusiasm in developing advanced super-resolution imaging and optical communications techniques. A compact VB nanolaser is intriguing for VB applications in miniaturized photonic integrated circuits. However, determined by the diffraction limit of light, it is a challenge to realize a VB nanolaser in the subwavelength scale because the VB lasing modes should have laterally structured distributions. Here, we demonstrate a VB nanolaser made from a 300 nm thick InGaAs/GaAs nanowire (NW). To select the high-order VB lasing mode, a standing NW as-grown from the selective-area-epitaxial (SAE) growth process is utilized, which has a bottom donut-shaped interface with the silicon oxide growth substrate. With this donut-shaped interface as one of the reflective mirrors of the nanolaser cavity, the VB lasing mode has the lowest threshold. Experimentally, a single-mode VB lasing mode with a donut-shaped amplitude and azimuthally cylindrical polarization distribution is obtained. Together with the high yield and uniformity of the SAE-grown NWs, our work provides a straightforward and scalable path toward cost-effective co-integration of VB nanolasers on potential photonic integrated circuits.

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

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

A Beginner’s Guide to Dye Selection and Labeling Strategies for Single-Molecule Localization Microscopy

AbstractSingle-molecule localization microscopy (SMLM) is an optical super-resolution technique that circumvents the diffraction limit of light by localizing the center point of each fluorophore’s recorded point spread function. SMLM requires the use of labeling strategies and specialized fluorophores and/or buffer conditions that enable most of the fluorophores in the sample to be “turned off” during the experiment so that a sparse subset of the fluorophores in the sample can be isolated from their nearest “on” neighbors and localized to build up an image of interest. Due to the nature of super-resolution single-molecule imaging, careful sample preparation, imaging buffer preparation, and optimized imaging conditions are fundamental requirements for obtaining quality data. This article discusses key factors to consider when preparing samples and acquiring data for SMLM to serve as a starting point for sample preparation and acquisition optimization.

Expansion microscopy through mechanical force.

In biophysics interesting phenomena take place during the interaction between biological molecules on the order of nanometers. Optical super-resolution imaging techniques surpass the diffraction limit of light enabling higher resolutions to study these phenomenon. However, super-resolution techniques usually require expensive equipment. Expansion microscopy (ExM) is a sample preparation method that facilitates super-resolution imaging by leveraging osmotic forces to physically separate features smaller than the diffraction limit of light. ExM is cheaper than and compatible with other super-resolution methods to reach the nanoscale. Currently, ExM commonly produces 4.5X linear expansion and 20X has been achieved using successive expansion steps, but this can result in fragmentation of the biological sample. Allowing the gel to absorb water to expand is also as low process. To overcome the fragmentation and time issues, here, we use a highly stretchable cross-linked polymer gel and a mechanical stretcher device to achieve similar 20X linear expansion in minutes. Since we do not rely on osmotic forces for the expansion, this method is more compatible with other techniques which are incompatible with excess water. We demonstrate our expansion microscopy method by imaging MOSE cells at 50 nm resolution. We establish mechanical force expansion microscopy as a viable sample preparation method with the same advantages as ExM while being much faster. This will also allow researchers to study time sensitive phenomenon.

Deep-learning applications in the automated image analysis of giant unilamellar vesicles.

Recent progress in the field of machine learning allowed scientists to develop software to analyze a great amount of image data efficiently. Trained artificial neural networks can segment, analyze, and classify the target objects in complex images. Face recognition technology in our cell phones is an everyday experience now. We have been developing automated image analysis software to analyze lipid vesicle images efficiently. Giant unilamellar vesicles (GUV), characterized by a diameter greater than 1 µm are routinely used for reconstitution studies. They are used to study a wide range of physiological activities involving lipid membranes. One of the great advantages of the GUV system is that they can be readily imaged, and studied by optical microscopy due to their size much greater than the resolution of visible light's diffraction limit. Fluorescence microscopy image analysis can reveal the protein-lipid interaction, phase separation on the membranes, and mechanical deformation of the membranes. Such analysis typically involves the manual analysis by researchers to identify the target vesicles to analyze and perform quantitative analysis. Our approach to introducing deep-learning can automate the process to help researchers to analyze a great amount of vesicle image data efficiently. Our work shows that with some training, scientists can introduce the deep-learning as a module for their image analysis for various purposes. We discuss the progress in the field of software analysis of vesicle images and show our example works of deep learning applications. Deep-learning assisted identification, and classification of vesicle states and their connection with quantitative analysis will be shown. We suggest practical ways to implement it for various purposes of image analysis. We envision such modules can be potentially introduced in the classroom to teach future scientists to learn machine learning applications in biophysics.

Estimating the localization spread function of static single-molecule localization microscopy images.

Single-molecule localization microscopy (SMLM) permits the visualization of cellular structures an order of magnitude smaller than the diffraction limit of visible light, and an accurate, objective evaluation of the resolution of an SMLM data set is an essential aspect of the image processing and analysis pipeline. Here, we present a simple method to estimate the localization spread function (LSF) of a static SMLM data set directly from acquired localizations, exploiting the correlated dynamics of individual emitters and properties of the pair autocorrelation function evaluated in both time and space. The method is demonstrated on simulated localizations, DNA origami rulers, and cellular structures labeled by dye-conjugated antibodies, DNA-PAINT, or fluorescent fusion proteins. We show that experimentally obtained images have LSFs that are broader than expected from the localization precision alone, due to additional uncertainty accrued when localizing molecules imaged over time.

Live-cell super-resolution imaging of actin using LifeAct-14 with a PAINT-based approach.

We present direct-LIVE-PAINT, an easy-to-implement approach for the nanoscopic imaging of protein structures in live cells using labeled binding peptides. We demonstrate the feasibility of direct-LIVE-PAINT with an actin-binding peptide fused to EGFP, the location of which can be accurately determined as it transiently binds to actin filaments. We show that direct-LIVE-PAINT can be used to image actin structures below the diffraction-limit of light and have used it to observe the dynamic nature of actin in live cells. We envisage a similar approach could be applied to imaging other proteins within live mammalian cells.

Near-field optical characterization of atomic structures and polaritons in twisted two-dimensional materials

Polariton is a quasiparticle generated from strong interaction between a photon and an electric or magnetic dipole-carrying excitation. These polaritons can confine light into a small space that is beyond the diffraction limit of light, thus have greatly advanced the development of nano photonics, nonlinear optics, quantum optics and other related research. Van der Waals two-dimensional (2D) crystals provide an ideal platform for studying nano-polaritons due to reduced material dimensionality. In particular, stacking and twisting offer additional degree of freedom for manipulating polaritons that are not available in a single-layer material. In this paper, we review the near-field optical characterizations of various structures and polaritonic properties of stacked/twisted 2D crystals reported in recent years, including domain structures of stacked few-layer graphene, moiré superlattice structures of twisted 2D crystals, twisted topological polaritons, and twisted chiral plasmons. We also propose several exciting directions for future study of polaritons in stacked/twisted 2D crystals.

STORM Microscopy and Cluster Analysis for PcG Studies.

Advanced microscopy techniques (such as STORM, STED, andSIM) have recently allowed the visualization of biological samples beyond the diffraction limit of light. Thanks to this breakthrough, the organization of molecules can be revealed within single cells as never before.Here, we describe the application of STochastic Optical Reconstruction Microscopy (STORM) for the study of polycomb group of proteins (PcG) in the context of chromatin organization. We present a clustering algorithm to quantitatively analyze the spatial distribution of nuclear molecules (e.g., EZH2 or its associated chromatin mark H3K27me3) imaged by 2D STORM. This distance-based analysis uses x-y coordinates of STORM localizations to group them into "clusters." Clusters are classified as singles if isolated or into islands if they form a group of closely associated clusters. For each cluster, the algorithm calculates the number of localizations, the area, and the distance to the closest cluster.This approach can be used for every type of adherent cell lineand allows the imaging of every protein for which an antibody is available. It represents a comprehensive strategy to visualize and quantify how PcG proteins and related histone marks organize in the nucleus at nanometric resolution.

LOV2-based photoactivatable CaMKII and its application to single synapses: Local Optogenetics.

Optogenetic techniques offer a high spatiotemporal resolution to manipulate cellular activity. For instance, Channelrhodopsin-2 with global light illumination is the most widely used to control neuronal activity at the cellular level. However, the cellular scale is much larger than the diffraction limit of light (<1 μm) and does not fully exploit the features of the "high spatial resolution" of optogenetics. For instance, until recently, there were no optogenetic methods to induce synaptic plasticity at the level of single synapses. To address this, we developed an optogenetic tool named photoactivatable CaMKII (paCaMKII) by fusing a light-sensitive domain (LOV2) to CaMKIIα, which is a protein abundantly expressed in neurons of the cerebrum and hippocampus and essential for synaptic plasticity. Combining photoactivatable CaMKII with two-photon excitation, we successfully activated it in single spines, inducing synaptic plasticity (long-term potentiation) in hippocampal neurons. We refer to this method as "Local Optogenetics", which involves the local activation of molecules and measurement of cellular responses. In this review, we will discuss the characteristics of LOV2, the recent development of its derivatives, and the development and application of paCaMKII.

Unbiased choice of global clustering parameters for single-molecule localization microscopy

Single-molecule localization microscopy resolves objects below the diffraction limit of light via sparse, stochastic detection of target molecules. Single molecules appear as clustered detection events after image reconstruction. However, identification of clusters of localizations is often complicated by the spatial proximity of target molecules and by background noise. Clustering results of existing algorithms often depend on user-generated training data or user-selected parameters, which can lead to unintentional clustering errors. Here we suggest an unbiased algorithm (FINDER) based on adaptive global parameter selection and demonstrate that the algorithm is robust to noise inclusion and target molecule density. We benchmarked FINDER against the most common density based clustering algorithms in test scenarios based on experimental datasets. We show that FINDER can keep the number of false positive inclusions low while also maintaining a low number of false negative detections in densely populated regions.

Hyperbolic metamaterial structures based on graphene for THz super-resolution imaging applications

The diffraction limit of light due to the loss of evanescent waves that carry high spatial frequency information in the far field restricts the practical applications of terahertz imaging technology. In order to break the diffraction limit, we investigate the super-resolution capability of different types of multilayered graphene-dielectric hyperbolic metamaterials. A super-resolution of λ/10 is achieved for both the cylindrical and planar structures. A prominent advantage of graphene-dielectric hyperbolic structures is the dynamic tunability of the dispersion and super-resolution performance by adjusting the chemical potential of graphene through conveniently changing the gate voltage without modifying the geometry of the hyperbolic structures. Furthermore, we have investigated the influence of bilayer thickness variations on the super-resolution performance. Finally, we apply the planar hyperbolic structures for the super-resolution imaging and a roughly five-fold lateral resolution enhancement is realized in our approach. Due to many prominent advantages including super-resolution over broad spectral range, dynamic tunability, good stability and robustness, we believe this work could contribute to the improvement of the resolutions of terahertz imaging systems and the development of hyperbolic metamaterial modulation devices in the terahertz band.

Extending resolution within a single imaging frame

The resolution of fluorescence microscopy images is limited by the physical properties of light. In the last decade, numerous super-resolution microscopy (SRM) approaches have been proposed to deal with such hindrance. Here we present Mean-Shift Super Resolution (MSSR), a new SRM algorithm based on the Mean Shift theory, which extends spatial resolution of single fluorescence images beyond the diffraction limit of light. MSSR works on low and high fluorophore densities, is not limited by the architecture of the optical setup and is applicable to single images as well as temporal series. The theoretical limit of spatial resolution, based on optimized real-world imaging conditions and analysis of temporal image stacks, has been measured to be 40 nm. Furthermore, MSSR has denoising capabilities that outperform other SRM approaches. Along with its wide accessibility, MSSR is a powerful, flexible, and generic tool for multidimensional and live cell imaging applications.

Hybrid Image Graphene Polaritons with Extremely Confined Mode and Field Enhancement in the Terahertz Regime

AbstractGuiding terahertz waves below the diffraction limit of light typically excite graphene plasmons (GPs). However, the mode confinements of conventional GPs using graphene–dielectric interfaces are limited. To further squeeze the optical mode sizes, a graphene–dielectric–metal (GDM) configuration supporting an image graphene plasmon (IGP) has been reported recently through the plasmon interactions in a graphene sheet and its mirror image inside a metal with out‐of‐phase charge oscillations. This study proposes a waveguide structure consisting of a cylindrical dielectric wire over a GDM to achieve an ultraconfined mode area by coupling a dielectric waveguide and an IGP mode. Furthermore, this work examines the dependence of waveguide dimensions and material properties on the mode characteristics of the proposed hybrid IGP (HIGP). This HIGP achieves plasmon wavelengths 20 times shorter and normalized propagation lengths twice longer than GPs, accompanying a mode size of 3 × 10−8A0, where A0 is the diffraction‐limited mode area. Moreover, the simple structure makes the HIGP easy to be verified experimentally. This work demonstrates that the HIGP delivering strong light–matter interactions at extremely compact dimensions can be exploited for building highly integrated, enhanced nonlinear, high‐resolution sensing and imaging THz devices.