Application Of Optical Microscopy Research Articles

Process development and validation of next generation 3D calibration standards for application in optical microscopy

Abstract A scalable wafer-based fabrication process for a new generation of 3D standards enabling the 3D calibration of optical microscopes is presented and validated. The 3D standards are based on step pyramids with several layers in the µm range and a system of cylindrical knops distributed across the layers as marks for coordinate based calibration. This enables calibration for the three coordinate axes and the orthogonality error between them in a single measurement step. The requirements necessary for such a calibration, as optical non-transparency, reproducible flatness of the pyramid step heights and the lowest possible deviations of the lateral marks coordinates, are met by optimizing the manufacturing process: The deviation of the height steps distributed over the wafer is ±3.6 nm and is primarily caused by the layer deposition processes. The lateral manufacturing accuracy was determined using calibrated scanning electron microscope (SEM) and show a mean deviation of 20 or 60 nm, depending on the lateral size of the structures. The electron beam lithography process and the level of inaccuracy of the SEM standard have an influence on the lateral scaling accuracy. Based on the tactilely generated height values and the coordinates of the mark determined by a calibrated SEM, an example calibration of a confocal laser scanning microscope was successfully performed and showed good conformity to conventional calibration techniques.

Self-referencing for quasi shot-noise-limited widefield transient microscopy

Many applications of ultrafast and nonlinear optical microscopy require the measurement of small differential signals over large fields-of-view. Widefield configurations drastically reduce the acquisition time; however, they suffer from the low frame rates of two-dimensional detectors, which limit the modulation frequency, making the measurement sensitive to excess laser noise. Here we introduce a self-referenced detection configuration for widefield differential imaging. Employing regions of the field of view with no differential signal as references, we cancel probe fluctuations and increase the signal-to-noise ratio by an order of magnitude reaching noise levels only a few percent above the shot noise limit. We anticipate broad applicability of our method to transient absorption, stimulated Raman scattering and photothermal-infrared microscopies.

Mechanism of Viral DNA Packaging in Phage T4 Using Single-Molecule Fluorescence Approaches.

In all tailed phages, the packaging of the double-stranded genome into the head by a terminase motor complex is an essential step in virion formation. Despite extensive research, there are still major gaps in the understanding of this highly dynamic process and the mechanisms responsible for DNA translocation. Over the last fifteen years, single-molecule fluorescence technologies have been applied to study viral nucleic acid packaging using the robust and flexible T4 in vitro packaging system in conjunction with genetic, biochemical, and structural analyses. In this review, we discuss the novel findings from these studies, including that the T4 genome was determined to be packaged as an elongated loop via the colocalization of dye-labeled DNA termini above the portal structure. Packaging efficiency of the TerL motor was shown to be inherently linked to substrate structure, with packaging stalling at DNA branches. The latter led to the design of multiple experiments whose results all support a proposed torsional compression translocation model to explain substrate packaging. Evidence of substrate compression was derived from FRET and/or smFRET measurements of stalled versus resolvase released dye-labeled Y-DNAs and other dye-labeled substrates relative to motor components. Additionally, active in vivo T4 TerS fluorescent fusion proteins facilitated the application of advanced super-resolution optical microscopy toward the visualization of the initiation of packaging. The formation of twin TerS ring complexes, each expected to be ~15 nm in diameter, supports a double protein ring-DNA synapsis model for the control of packaging initiation, a model that may help explain the variety of ring structures reported among pac site phages. The examination of the dynamics of the T4 packaging motor at the single-molecule level in these studies demonstrates the value of state-of-the-art fluorescent tools for future studies of complex viral replication mechanisms.

Generation of spatial combs digitized by orbital angular momentum

Shaping the transverse spatial domain of light has been experiencing recent growth in attention for its applications in optical tweezing, microscopy, communications, and quantum information sciences. The orbital angular momentum (OAM) of light is a transverse physical property that functions as a viable basis for many of such applications. While fields containing a single OAM mode order have extensively been used, fields containing a broad, discrete, and equally spaced OAM spectrum—an OAM comb—has largely been unexplored due to a lack of viable creation methods. Much like how frequency combs enabled myriad applications, it is conceivable that OAM combs would likewise enable further opportunities or expand upon single-OAM applications. For instance, an OAM comb may pave the way for more sophisticated particle manipulation, object detection, and pump shaping for high-dimensional spatial-mode entanglement. Here, we create four OAM combs with rectangular, sinusoidal, sinc, and Gaussian OAM spectral distributions using multiplane light conversion, which only involves phase modulations (hence is inherently lossless) and allows for improved tailoring of the spatial shape. The verification of such combs is done qualitatively by analyzing their propagation evolutions and quantitatively by employing a mode sorting technique—both exhibiting good agreement with user specifications and simulations. The scattering losses of all four combs were measured to be minimal, ranging from 0.66 to 1.04 dB.

Adaptive optics for optical microscopy [Invited

Optical microscopy is widely used to visualize fine structures. When applied to bioimaging, its performance is often degraded by sample-induced aberrations. In recent years, adaptive optics (AO), originally developed to correct for atmosphere-associated aberrations, has been applied to a wide range of microscopy modalities, enabling high- or super-resolution imaging of biological structure and function in complex tissues. Here, we review classic and recently developed AO techniques and their applications in optical microscopy.

Observation of Zn Dendrite Growth via Operando Digital Microscopy and Time-Lapse Tomography.

The zinc-ion battery is one of the promising candidates for next-generation energy storage devices beyond lithium technology due to the earth's abundance of Zn materials and their high volumetric energy density (5855 mA h cm-3). To date, the formation of Zn dendrites during charge-discharge cycling still hinders the practical application of zinc-ion batteries. It is, therefore, crucial to understand the formation mechanism of the zinc dendritic structure before effectively suppressing its growth. Here, the application of operando digital optical microscopy and in situ lab-based X-ray computed tomography (X-ray CT) is demonstrated to probe and quantify the morphologies of zinc electrodeposition/dissolution under multiple galvanostatic plating/stripping conditions in symmetric Zn||Zn cells. With the combined microscopy approaches, we directly observed the dynamic nucleation and subsequent growth of Zn deposits, the heterogeneous transportation of charged clusters/particles, and the evolution of 'dead' Zn particles via partial dissolution. Zn electrodeposition at the early stage is mainly attributed to activation, while the subsequent dendrite growth is driven by diffusion. The high current not only facilitates the formation of sharp dendrites with a larger mean curvature at their tips but also leads to dendritic tip splitting and the creation of a hyper-branching morphology. This approach offers a direct opportunity to characterize dendrite formation in batteries with a metal anode in the laboratory.

Study on the application of optical coherence microscopy in Hirschsprung's disease

To explore the clinical application value of optical coherence microscopy (OCM) in Hirschsprung’s disease. 109 HSCR patients were recuited in a Chinese hospital from January 2018 to July 2021. All the recruited patients underwent barium enema angiography preoperatively and the resected diseased intestinal tubes were evaluated intraoperatively. The OCM and the histopathological examination were performed successively on the surgical specimens, and the OCM images were compared with the relevant tissue sections to characterize different lesions. 10 non-HSCR fetal colorectal tissues at the same period were retained for OCM, the characteristics of which with and without HSCR under OCM imaging were analyzed. In the OCM images of in vitro tissue, it can be clearly observed that the scattering degree of HSCR narrow segment mucosal is high, glands and crypt structures are reduced or even atrophy, and the scattering degree of submucosal and intermuscular is low; In the dilated segment, the low scattering and high scattering are complex, and the muscle layer is obviously hypertrophy and structural disorder. Compared with the pathological findings, the OCM sensitivity, Kappa value, and AUC area reached 92.66%, 0.63, and 0.91, respectively. OCM can quickly and clearly display the structure of all layers of colorectal tissue, which is highly consistent with the corresponding histopathological examination results and has high sensitivity. which will provide a more reliable basis for OCM diagnosis of early HSCR, targeted biopsy and location of operative treatment, and has a certain potential for clinical application.

Antibody Functionalization of Ultrasmall Fluorescent Core–Shell Aluminosilicate Nanoparticle Probes for Advanced Intracellular Labeling and Optical Super Resolution Microscopy

Fluorescent labeling of cellular substructures is commonly performed using antibody–organic dye conjugates. Organic dyes do not exhibit ideal optical properties in terms of brightness and photostability, however, in particular when it comes to advanced optical super-resolution microscopy (SRM) applications. Here, we demonstrate the efficient conjugation of widely available secondary antibodies and cationic species to ultrasmall (sub-10 nm) fluorescent silica core─poly(ethylene glycol) shell (core–shell) aluminosilicate nanoparticles (aC′ dots) encapsulating different color dyes for specific targeting and high-quality fluorescence imaging of structures of the cytoskeleton (tubulin and actin) and nucleus, respectively. We show that the different color aC′ dots provide enhanced brightness and photostability relative to their parent dyes. As recently discovered, we further demonstrate that they exhibit photo-induced blinking with low ON–OFF duty cycles enabling optical SRM, for example, in the form of stochastic optical reconstruction microscopy (STORM), without the need for complex imaging setups or cocktails. After carefully optimizing Ab–aC′ dot conjugation as well as cell structure labeling protocols in fixed and permeabilized HeLa and MDA-MB-231 cells, we demonstrate three-color STORM and exemplify improved resolution compared to standard antibody–dye conjugates. This work paves the way to next-generation multifunctional optical probes based on ultrasmall silica nanoparticle platforms for advanced applications in bioimaging, nanomedicine, and beyond.

Generating Ultralong-Superoscillation Nondiffracting Beams with Full Control of the Intensity Profile

Nondiffracting light beams receive substantial attention in various areas due to their properties of diffractionless propagation and achievable subwavelength beam size. Extending the propagation distance and compressing the beam size of nondiffracting beams are crucial to their practical applications. However, it remains challenging to achieve nondiffracting light beams with a subwavelength subdiffracting transverse size and a long propagation distance with controllable intensity profiles in both transverse and longitudinal directions. Here, we propose an optimization method to generate an ultralong-superoscillation nondiffracting light beam with full control of the beam intensity profile. A 45\ensuremath{\lambda}-long-superoscillation nondiffracting light beam, the transverse sizes of which are within the range of 0.37\ensuremath{\lambda}--0.4\ensuremath{\lambda} along the propagation axis, is experimentally demonstrated by a metalens based on continuous phase modulation using geometrical phase metasurfaces. Our method provides an attractive way to controllably generate ultralong-superoscillation nondiffracting beams on a subwavelength scale and might find fascinating applications in optical manipulation, materials processing, data storage, microscopy, and spectroscopy.

Sortase A transpeptidation produces seamless, unbranched biotinylated nanobodies for multivalent and multifunctional applications.

Exploitation of the biotin-streptavidin interaction for advanced protein engineering is used in many bio-nanotechnology applications. As such, researchers have used diverse techniques involving chemical and enzyme reactions to conjugate biotin to biomolecules of interest for subsequent docking onto streptavidin-associated molecules. Unfortunately, the biotin-streptavidin interaction is susceptible to steric hindrance and conformational malformation, leading to random orientations that ultimately impair the function of the displayed biomolecule. To minimize steric conflicts, we employ sortase A transpeptidation to produce quantitative, seamless, and unbranched nanobody-biotin conjugates for efficient display on streptavidin-associated nanoparticles. We further characterize the protein-nanoparticle complex and demonstrate its usefulness in optical microscopy and multivalent severe acute respiratory syndrome coronavirus (SARS-CoV-2) antigen interaction. The approach reported here provides a template for making novel multivalent and multifunctional protein complexes for avidity-inspired technologies.

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.

Fluorescence microscopy imaging of a neurotransmitter receptor and its cell membrane lipid milieu.

Hampered by the diffraction phenomenon, as expressed in 1873 by Abbe, applications of optical microscopy to image biological structures were for a long time limited to resolutions above the ∼200nm barrier and restricted to the observation of stained specimens. The introduction of fluorescence was a game changer, and since its inception it became the gold standard technique in biological microscopy. The plasma membrane is a tenuous envelope of 4nm-10nm in thickness surrounding the cell. Because of its highly versatile spectroscopic properties and availability of suitable instrumentation, fluorescence techniques epitomize the current approach to study this delicate structure and its molecular constituents. The wide spectral range covered by fluorescence, intimately linked to the availability of appropriate intrinsic and extrinsic probes, provides the ability to dissect membrane constituents at the molecular scale in the spatial domain. In addition, the time resolution capabilities of fluorescence methods provide complementary high precision for studying the behavior of membrane molecules in the time domain. This review illustrates the value of various fluorescence techniques to extract information on the topography and motion of plasma membrane receptors. To this end I resort to a paradigmatic membrane-bound neurotransmitter receptor, the nicotinic acetylcholine receptor (nAChR). The structural and dynamic picture emerging from studies of this prototypic pentameric ligand-gated ion channel can be extrapolated not only to other members of this superfamily of ion channels but to other membrane-bound proteins. I also briefly discuss the various emerging techniques in the field of biomembrane labeling with new organic chemistry strategies oriented to applications in fluorescence nanoscopy, the form of fluorescence microscopy that is expanding the depth and scope of interrogation of membrane-associated phenomena.

Microsphere assistance in interference microscopy with high numerical aperture objective lenses

Various attempts have been discussed to overcome the lateral resolution limit and thus to enlarge the fields of application of optical interference microscopy. Microsphere-assisted microscopy and interferometry have proven that the imaging of structures well below Abbe’s resolution limit through near-field assistance is possible if microspheres are placed on the measured surface and utilized as near-field assisting imaging elements. The enhancement of the numerical aperture (NA) by the microspheres as well as photonic nanojets was identified to explain the resolution enhancement, but also whispering gallery modes and evanescent waves are assumed to have an influence. Up to now, to the best of our knowledge, there is no complete understanding of the underlying mechanisms and no model enabling to examine ideal imaging parameters. This contribution is intended to clarify how much the lateral resolution of an already highly resolving Linnik interferometer equipped with 100 × NA 0.9 objective lenses can be further improved by microspheres. Our simulation model developed so far is based on rigorous near-field calculations combined with the diffraction-limited illumination and imaging process in an interference microscope. Here, we extend the model with respect to microsphere-assisted interference microscopy providing a rigorous simulation of the scattered electric field directly above the sphere. Simulation and experimental results will be compared in the three-dimensional spatial frequency domain and discussed in context with ray-tracing computations to achieve an in-depth understanding of the underlying mechanism of resolution enhancement by the microsphere.

Strongly coupled surface plasmon resonance modes at both FF and SH in clusters of symmetrically distributed metallic nanospheres

Plasmonic nanoclusters play an important role in the rapid development of application of nanophotonics. In this article both the fundamental-frequency (FF) and second-harmonic (SH) responses of the complex nanoclusters (tetramer, hexamer, and octamer), constructed by coupled spherical plasmonic nanoparticles, are theoretically investigated. By using the multiple scattering algorithm, we present the comprehensive numerical analysis of the physical mechanisms pertaining to the linear and nonlinear coupled-plasmon modes, including ‘hot spot’ modes (HSMs) and ‘whispering-gallery’ modes (WGMs). Different from the symmetric coupling of plasmonic eigenmodes excited by the nanodimer, our analysis reveals that such coupled modes from the nanomultimers are the result of the ‘hybridization’ of symmetric and asymmetric coupling between adjacent dipole or higher-order multipole moments. Also, the remarkable enhancement as large as 3–4 orders of magnitude is achieved for the SH intensity when employing the plasmonic HSMs or WGMs at the FF. Interestingly, the nonlinear plasmonic HSMs and WGMs (the maximum Q up to 4800) can also be obtained by elaborately designing the system geometry. In addition, the resonant wavelengths of such coupled-plasmon modes can be linearly modulated within a broad range. These numerical results have potential applications in surface optical microscopy, plasmonic sensor and nanolaser.

The northern-Italy Renaissance in a panel by Defendente Ferrari: A complete study with a multi-analytical investigation

Defendente Ferrari, one of the most important painters of the Piedmontese Renaissance, was extremely active in the early 16th century. His Madonna with Child and two Saints (1505–1510 ca.), which was originally located in the Collegiate Church of Carmagnola (Turin province, Italy) now in the collections of Palazzo Madama - Museo Civico d’Arte Antica in Turin, was recently analyzed and restored at the Centro Conservazione e Restauro La Venaria Reale, as part of a study and conservation project focused on the artist and his circle.This study presents an interdisciplinary and multi-analytical archaeometric approach resulting from the collaboration of several universities and research centers. The adopted protocol allowed for clarifying some peculiarities of the artist's painting technique, such as the use of the dotted - a technique applied to render the nuances, volume, and flesh tones of the figures - and that of thin black cross-hatched strokes, drawn to shape the clothes and to define the related volumes and folds. Moreover, specific degradation processes were studied, which apparently occur also in other contemporary artworks with similar features. Of particular interest is the alteration of the blue color of the Virgin's garment, made of azurite and presently strongly blackened.SWIR reflectography performed with a high-resolution InGaAs scanner prototype allowed for imaging both the detailed underdrawing and the careful hatching used to highlight the shadows. The combined application of optical microscopy, SEM-EDS, µ-Raman, FT-IR spectroscopy, and GC–MS has been fundamental for analyzing some specific pigments, binders, and varnishes, as well as for inferring the cause for Virgin's garment blackening. A complete mapping of the pigments was provided through the combination of FORS (350–2000 nm), portable µ-Raman, MA-XRF, and Hypercolorimetric Multispectral Imaging. The latter also allowed for observing the distribution of what is supposed to be either the original or the ancient organic finishing layer, which was maintained during the conservation treatment.This research has brought to an in-depth knowledge of the artist's painting technique and helped the adoption of the best choices for an effective and minimal-invasive restoration intervention.

Correlated Study of Material Interaction Between Capillary Printed Eutectic Gallium Alloys and Gold Electrodes.

Liquid metals (LMs) play a growing role in flexible electronics and connected applications. Here, LMs come into direct contact with metal electrodes thus allowing for corrosion and additional alloying, potentially compromising device stability. Nevertheless, comprehensive studies on the interfacial interaction of the materials are still sparse. Therefore, a correlated material interaction study of capillary-printed Galinstan (eutetic alloy of Ga/In/Sn) with gold surfaces and electrodes is conducted. Comprehensive application of optical microscopy, vertical scanning interferometry, scanning electron microscopy/spectroscopy, x-ray photon spectroscopy, and atomic force microscopy allow for an in depth characterization of the spreading process of LM lines on gold films, revealing the differential spread of the different LM components and the formation of intermetallic nanostructures on the surface of the surrounding gold film. A model for the growth process based on the penetration of LM along the gold film grain boundaries is proposed based on the obtained time-dependent characterization. The distribution of gold, Galinstan, and intermetallic phases in a gold wire dipped into LM is observed using X-ray nano tomography as a complementary view on the internal nanostructure. Finally, resistance measurements on LM lines connecting gold electrodes over time allow to estimate the influence on the material interaction on electronic applications.

Toward High‐Efficiency Ultrahigh Numerical Aperture Freeform Metalens: From Vector Diffraction Theory to Topology Optimization

AbstractOptical metasurface is a 2D array of subwavelength meta‐atoms that arbitrarily manipulates amplitude, polarization, and wavefront of incident light, offering great advantages in miniaturization and integration. However, the presuppositions hidden in conventional unit‐cell‐based design approach, including discrete phase sampling, local periodicity approximation, and normal response, imply that the responses and interactions of meta‐atoms are almost impossible to be accurately modeled, thus impeding the effective design of high‐efficiency ultrahigh numerical aperture (NA) metalens. Here, based on vector diffraction theory and plane wave expansion method, theoretical limitation of metalens efficiency is comprehensively investigated. It is identified that for high‐NA metalens, theoretical focusing efficiency is limited by diffraction capability of vector field, while evanescent wave attenuation dominates theoretical efficiency decline for ultrahigh‐NA metalens. It is also shown that the efficiency of conventional metalens has a huge gap from theoretical limitation, owing to imperfect diffractive focusing and impedance mismatch reflections. To fill such efficiency gap, the high‐efficiency high‐NA freeform metalens based on topology optimization is further demonstrated. Particularly, topology geometric constraints are utilized to reduce minimum feature size while keeping high efficiency. The results could shed new light on the understanding and design of ultrahigh‐NA metalens and find promising applications in optical imaging, microscopy, and lithography.

Tight focusing of high-order polarized anomalous vortex beam

Based on the vector Debye theory, the tight focusing properties of a high-order polarized anomalous vortex (HPAV) beam are studied. The corresponding mathematical expressions of the HPAV beam are derived theoretically. We accomplish the inner and outer gear shapes of the focusing intensity where the number of the gear tooth can be modulated by polarization order. The results show that the focusing gear intensity can be flexibly modulated by initial polarization azimuth which may determine the trapping effects. Various charming focusing field patterns can be used to capture two kinds of different refractive indices particles simultaneously. The compactness of the intensity distribution can be freely adjusted by the HPAV beam topological charges and polarization order. The focal spot size, which is far beyond the Rayleigh diffraction limitation can be achieved. It may be expected to have potential applications in optical microscopy, imaging, optical telecommunication and other fields.

Focusing of radially polarized bessel gaussian and hollow gaussian beam of high NA to achieve super resolution

To achieve super-resolution, a radially polarized Bessel Gaussian and a Hollow Gaussian Beam with high NA are focused. For both Bessel Gaussian and Hollow Gaussian Beams with high numerical aperture NA, the radially polarized component with two equal-intensity peaks covers 25% of the total intensity, while the longitudinally polarized component covers the rest. By focusing radially polarized Bessel and Hollow Gaussian beams, it is possible to generate various focal spots with different dimensions that are applicable for various applications in optical trapping, data storage, biomedical imaging, laser cutting, material processing, and microscopy. The effects of various Bessel beam characteristics are investigated. It was found that the beam radius reaches a minimum at a specified starting beam radius for all propagation distances and that the optimum starting beam radius is also large at long propagation distances. This findings are of great significance in optical trapping, data storage, biomedical imaging, and laser cutting.

Orbital angular momentum and beyond in free-space optical communications

Abstract Orbital angular momentum (OAM), which describes tailoring the spatial physical dimension of light waves into a helical phase structure, has given rise to many applications in optical manipulation, microscopy, imaging, metrology, sensing, quantum science, and optical communications. Light beams carrying OAM feature two distinct characteristics, i.e., inherent orthogonality and unbounded states in principle, which are suitable for capacity scaling of optical communications. In this paper, we give an overview of OAM and beyond in free-space optical communications. The fundamentals of OAM, concept of optical communications using OAM, OAM modulation (OAM modulation based on spatial light modulator, high-speed OAM modulation, spatial array modulation), OAM multiplexing (spectrally efficient, high capacity, long distance), OAM multicasting (adaptive multicasting, N-dimensional multicasting), OAM communications in turbulence (adaptive optics, digital signal processing, auto-alignment system), structured light communications beyond OAM (Bessel beams, Airy beams, vector beams), diverse and robust communications using OAM and beyond (multiple scenes, turbulence-resilient communications, intelligent communications) are comprehensively reviewed. The prospects and challenges of optical communications using OAM and beyond are also discussed at the end. In the future, there will be more opportunities in exploiting extensive advanced applications from OAM beams to more general structured light.