Reflection Microscopy Research Articles

Measurements and simulations of microtubule growth imply strong longitudinal interactions and reveal a role for GDP on the elongating end.

Microtubule polymerization dynamics result from the biochemical interactions of αβ-tubulin with the polymer end, but a quantitative understanding has been challenging to establish. We used interference reflection microscopy to make improved measurements of microtubule growth rates and growth fluctuations in the presence and absence of GTP hydrolysis. In the absence of GTP hydrolysis, microtubules grew steadily with very low fluctuations. These data were best described by a computational model implementing slow assembly kinetics, such that the rate of microtubule elongation is primarily limited by the rate of αβ-tubulin associations. With GTPase present, microtubules displayed substantially larger growth fluctuations than expected based on the no GTPase measurements. Our modeling showed that these larger fluctuations occurred because exposure of GDP-tubulin on the microtubule end transiently 'poisoned' growth, yielding a wider range of growth rates compared to GTP only conditions. Our experiments and modeling point to slow association kinetics (strong longitudinal interactions), such that drugs and regulatory proteins that alter microtubule dynamics could do so by modulating either the association or dissociation rate of tubulin from the microtubule tip. By causing slower growth, exposure of GDP-tubulin at the growing microtubule end may be an important early event determining catastrophe.

Uncovering the physical properties, structural characteristics, and electronic application of superlattice-like Ti/Sb phase-change thin films

Superlattice-like (SLL) Ti/Sb thin films were proposed and investigated from the viewpoint of physical properties, structural characteristics, and electronic application. Magnetron sputtering was employed to deposit the SLL Ti/Sb thin films with different thickness ratios. In-situ resistance–temperature measurement indicates that the crystallization temperature, crystallization-activation energy, and data-retention capacity increase significantly and the resistance drift index reduces with an increment in thickness ratio of the Ti to Sb layer, meaning higher amorphous thermal stability and reliability of SLL Ti/Sb thin films. X-ray diffraction and Raman spectra reveal that the inserted Ti layer can inhibit grain growth and refine the grain size, causing remarkable improvement of thermal stability and crystalline resistance. Analyses of x-ray reflectivity and atomic force microscopy demonstrate that the thickness fluctuation of SLL Ti/Sb thin films becomes smaller and the surface topography becomes smoother, respectively. The Avrami exponent of the SLL (Ti3Sb7)5 thin film reflects the growth-dominated crystallization mechanism, implying a rapid phase transition speed. Phase-change memory cells based on the SLL (Ti3Sb7)5 thin film can realize a reversible SET/RESET operation under an electrical pulse with a width of 100 ns. The RESET power consumption was estimated to be much lower than that of traditional Ge2Sb2Te5 material. The above results strongly prove that the suitable SLL structure of Ti/Sb thin films have tremendous potential in the area of high-temperature and low-power electronic storage.

A physics-based model for crystal orientation dictionary indexing by directional reflectance microscopy

Directional reflectance microscopy (DRM) is a new optical technique that enables grain orientation mapping in crystalline solids by capturing and analyzing light reflectance signals generated by chemically etched surfaces. Currently, orientation indexing by DRM relies on fitting the optical signals to identify user-defined features that carry orientation information. This approach is inevitably error-prone and material-dependent. These shortcomings hinder the adoption of DRM as a universal characterization method in materials science. We propose a new indexing method to improve the robustness and versatility of DRM. Our method relies on building a dictionary of all possible reflectance signals generated by a metal, which we simulate using a physics-based forward model that takes crystal orientation as input. We then compare each measured reflectance signal acquired by DRM to all the entries in the dictionary in search of the best match, and thus the correct crystal orientation. We demonstrate our dictionary indexing DRM (DI-DRM) approach on nickel and aluminum polycrystals, which produce markedly different optical reflectance signals. We find that DI-DRM yields measurements with improved accuracy compared to those enabled by fitting the optical signal on both materials and across all crystal orientations considered. We also show that the measurement error (∼3°) is mildly sensitive to experimental variability, including noise, measurement settings, and sample surface preparation. DI-DRM represents a considerable step forward towards the implementation of DRM as a streamline materials characterization technique.

Enhancing near-field optical tweezers by spin-to-orbital angular momentum conversion

Near-field patterns of light provide a way to optically trap, deliver, and sort single nanoscopic particles in a wide variety of applications in nanophotonics, microbiology, and nanotechnology. Using rigorous electromagnetic theory, we investigate the forces and trapping performance of near-field optical tweezers carrying spin and orbital angular momenta. The trapping field is assumed to be generated by a total internal reflection microscope objective at a glass–water interface in conditions where most of the transmitted light is evanescent. We discover aspects of these tweezers, including the possibility to rotate and stably trap nanoscopic beads. More importantly, we show that, under near-field conditions, the contributions of spin and orbital angular momenta to the rotation of small particles are almost equivalent, opening the possibility to cancel each other when they have an opposite sign. We show that these conditions result in optimal optical trapping, giving rise to extremely effective optical tweezers for nanomanipulation, with both circular symmetry and relatively weak rotation.

A mussel-bioinspired coating strategy to licence PCL osteoinductive activity for repairing bone defects

3,4-Dihydroxyphenylalanine (DOPA) is believed to play an essential role in the solidification and adhesion of liquid mussel adhesives; this thin polymer layer very tightly adheres to the surface of scaffolds, making it stable enough to prevent it from being peeled off. Fascinatingly, the deposition of DOPA on various types of surfaces via the self-polymerization of DOPA under alkaline conditions is a simple approach for creating biomimetic coatings. In this study, DOPA derivatives were used as monomers to form primary coatings on polycaprolactone (PCL) surfaces. After that, a second chemical crosslinking method was utilized to graft alendronate (AL) on the DOPA coatings via genipin (GP) crosslinking (D-PCL@AL). X-ray photoelectron spectroscopy, Fourier transform infrared reflection and scanning electron microscopy analyses indicated that AL was distributed homogeneously on the surface of PCL. This new organic–inorganic hybridized coating could support EMSC growth and osteogenic differentiation. Additionally, animal studies were also performed to verify the function of D-PCL@AL in promoting osteogenesis. The method is simple, cheap and highly efficient, which makes it very convenient to apply on a large scale.

Dynamic, Spontaneous Blistering of Substrate-Supported Graphene in Acidic Solutions.

We report that for monolayer and few-layer graphene on common silicon and glass substrates, acidic solutions induce fast, spontaneous generation of solution-enclosing blisters/bubbles. Using interference reflection microscopy, we monitor the blister-generating process in situ and show that at pH < ∼2, nanoscale to micrometer-sized graphene blisters, up to ∼100 nm in height, are universally generated with high surface coverages on hydrophilic, but not hydrophobic, surfaces. The spontaneously generated blisters are highly dynamic, with growth, merging, and reconfiguration occurring at second-to-minute time scales. Moreover, we show that in this dynamic system, graphene behaves as a semipermeable membrane that allows the relatively free passing of water, impeded passing of the NaCl solute, and no passing of large dye molecules. Consequently, the blister volumes can be fast and reversibly modulated by the solution osmotic pressure.

Background Pressure Induced Structural and Chemical Change in NiV/B4C Multilayers Prepared by Magnetron Sputtering

NiV/B4C multilayers with a small d-spacing are suitable for multilayer monochromator working at a photon energy region from 5 to 8 keV, or photon energy region from 10 to 100 keV. To investigate the influence of background pressure during fabrication, NiV/B4C multilayers with a d-spacing of 3.0 nm were fabricated by magnetron sputtering with different background pressures. The grazing incidence x-ray reflectivity (GIXR) and transmission electron microscopy (TEM) measurement illustrated the structural change that happened in NiV/B4C multilayers when background pressure is high. The electron dispersive x-ray spectroscopy (EDX) of NiV/B4C multilayer deposited with a high background pressure suggests a gradient distribution of oxygen, which corresponds to the gradient thickness change. The detailed x-ray absorption near edge spectroscopy (XANES) comparison of NiV/B4C multilayers, NiV coating, and B4C coating showed the chemical state change induced by background pressure. We concluded that during the deposition, vanadium oxide promoted the oxidation of boron. In order to fabricate a good performance of NiV/B4C multilayers, the background pressure needs lower than 1 × 10−4 Pa.

Effect of heterogeneous substrate adhesivity of follower cells on speed and tension profile of leader cells in primary keratocyte collective cell migration.

ABSTRACTIn single keratocyte motility, membrane tension is reported to be high at cell-fronts and believed to establish front coherence. To understand role of membrane mechanics in collective cell migration, we study membrane height fluctuations in cell sheets from fish scales using interference reflection microscopy (IRM). We report the monolayer to have cells lacking substrate adhesion and show that such ‘non-sticky’ cells can form bridges between leader cells and far-away follower cells. Do such interactions alter motility and membrane mechanics in such leaders? We find non-significant, but reduced speed for leaders with ‘non-sticky’ followers in comparison to other leaders. Cells show high phenotypic variability in their membrane fluctuation tension profiles. On average, this tension is found to be lower at cell fronts than the mid-section. However, leaders with non-sticky followers are more prone to display higher tension at their front and have a negative correlation between cell speed and front-mid tension difference. Thus, we conclude that intracellular tension gradients are heterogeneous in cell sheets and substrate adhesivity of followers can control the coupling of the gradient to cell speed.

Maxwell-Wagner Relaxation-Driven High Dielectric Constant in Al2O3/TiO2 Nanolaminates Grown by Pulsed Laser Deposition.

Multilayer nanolaminates (NLs) of alternate ultrathin sublayers of Al2O3 and TiO2 (ATA) with the thickness ranging ∼2 to 0.5 nm were fabricated by optimized pulsed laser deposition (PLD). Maxwell-Wagner (M-W) relaxation-induced interfacial polarization was realized and engineered by precisely controlling the sublayer thicknesses and the number of interfaces. X-ray reflectivity and cross-sectional transmission electron microscopy measurements of ATA NLs revealed an artificial periodicity with well-defined uniformly thick amorphous sublayers with chemically and physically distinct interfaces down to a sublayer thickness of ∼0.8 nm. The dielectric constants and loss of ATA NLs were found to increase from ∼60 to 670 and decrease from ∼0.9 to 0.16, respectively, as sublayer thicknesses reduced from ∼2 to 0.8 nm. However, for a sublayer thickness below 0.8 nm, the trend was reversed. Furthermore, temperature-dependent impedance spectroscopy studies revealed two distinct thermally activated relaxation processes, corresponding to TiO2 and Al2O3 sublayers, corroborating the M-W relaxation. The conductivity contrast between the sublayers of ATA NLs enhanced with reducing sublayer thickness and plateaued at a sublayer thickness of ∼0.8 nm, resulting in dominant M-W interfacial polarization and a high cut-off frequency of ∼50 kHz. These results demonstrate that ATA NLs grown by PLD may find application as potential high-k materials for next-generation nanoelectronic devices.

Dermoscopy and Reflectance Confocal Microscopy in Actinic Keratosis, Intraepithelial Carcinoma, and Invasive Squamous Cell Carcinoma.

Patients with multiple actinic keratosis (AK), have pre-neosplastic abnormalities, constituting the sites of new tumors, this region is called the cancerization field. Due to the risk of malignant transformation, rigorous evaluation, follow-up, and treatment of the cancerization field is proposed. Recently, non-invasive diagnostic technologies such as confocal reflectance microscopy (RCM), detect AK, intraepithelial carcinomas (IEC), and SCC, without the need of repeated biopsies. There are few reports of the progression of AK assessed by dermatoscopy and RCM concomitantly. Define morphological patterns and clinical applicability of dermatoscopy and MCR examinations of the AK lesions and their degrees of progression to IEC and SCC. A retrospective cross-sectional study of dermatoscopy and RCM examinations was performed in 30 patients with histopathological diagnosis of AK (20), IEC (6), and SCC (4). In the comparative analysis of the dermatoscopic features, erythema was present in 100% of the lesions, the red pseudo-network in 75% of the AK (P=0.007), and linear and irregular vessels in 90% of the lesions of IEC/SCC. In the RCM of AK, the most striking finding was the presence of atypical honeycomb in the spinous layer, but typical in the granular layer. While the IEC/SCC group presented irregular epidermal architecture and atypical honeycomb in all epider-mal layers, it also showed a higher prevalence of individual corneocytes and nucleated cells, cellular pleomorphism, and nuclear atypia in the dermal papillae, irregular vessels within papilla, and cells with bright edges and dark central nuclei in the dermis. Dermoscopy and RCM may be considered as auxiliary methods for assessing lesions resulting from ke-ratinocyte atypia. The results of this study are consistent with published studies and it was possible to propose, with literature support, a model of progression of AK to IEC and SCC. J Drugs Dermatol. 2022;21(3):259-268. doi:10.36849/JDD.5086.

High-throughput microstructure and composition characterisation of microplatelet reinforced composites using directional reflectance microscopy

Reinforced composites with controlled microstructure can achieve exceptional mechanical and functional properties. In recent years, scientists have placed in substantial efforts to fabricate reinforced composites with astounding properties through the design of these microstructures. To better understand the structure-property relationships of these materials, it is essential to characterise their microstructural features. However, standard characterisation techniques such as electron-based imaging are costly, time-consuming, and restricted to small-scale samples. In this work, we present a simple, inexpensive, and high-throughput alternative method to assess the surface microstructure of microplatelet reinforced composites. The method relies on directional reflectance microscopy (DRM), an optical technique that enables mapping of the filler orientation and weight fraction in these composites using surface reflectance data. We demonstrate the effective use of DRM on reflective microplatelet-reinforced polymeric composites that we prepared using 3D printing and mould casting techniques. Our experimental results showcase the potential of DRM as a high-throughput method to assess microstructural information in reinforced composites. This information could be used to understand how the microplatelets’ alignments influence the materials’ properties for devising new composite designs.

Encoding data into metal alloys using laser powder bed fusion

Beyond near-net shape manufacturing of parts with complex geometry, additive manufacturing (AM) makes it possible to fabricate materials with distinct, site-specific microstructures. This ability is unique to AM and enables the design of architected materials that were previously unattainable. Here, we leverage this strategy to encode data into metal parts using the microstructure as the medium to store information. We use a novel laser scanning technique to control the local solidification conditions during laser powder bed fusion and embed a linear barcode and Quick Response (QR) code into stainless steel 316 L. The codes use blocks of different crystallographic texture–i.e., regions in which the crystal lattice of most grains is aligned towards a preferred orientation–as the data “bits”. The data may be retrieved by analytical techniques that are sensitive to local microstructure variations. As a demonstration, we decode the barcodes by measuring the scattering of optical light from their etched surface using a technique called directional reflectance microscopy. The resulting texture maps are readable by conventional barcode scanners, such as those found on mobile phones. The ability to embed data has significant potential in fields such as law enforcement, biomedicine, and transportation, where permanent, damage-resistant tracking is essential.

Confocal reflectance microscopy for metal and lipid nanoparticle visualization in the brain.

Aim: A requirement for nanoparticle (NP) research is visualization of particles within cells and tissues. Limitations of electron microscopy and low yields of NP fluorescent tagging warrant the identification of alternative imaging techniques. Method: Confocal reflectance microscopy (CRM) in combination with fluorescence imaging was assessed for visualizing rhodamine B-conjugated silver and fluorescein isothiocyanate-conjugated lipid core-stearylamine NP uptake in vitro and in vivo. Results: CRM successfully identified cellular uptake and blood-brain barrier penetration of NPs owing to their distinguishing refractive indices. NP-dependent reflectance signals in vitro were dose and incubation time dependent. Finally, CRM facilitated the distinction between nonspecific fluorescence signals and NPs. Conclusion: These findings demonstrate the value of CRM for NP visualization in tissues, which can be performed with a standard confocal microscope.

Dimensional reduction for single-molecule imaging of condensation events

Macromolecules organize themselves into discrete membrane-less compartments through a process called liquid-liquid phase separation (LLPS). Mounting evidence has suggested that nucleosomes as well as DNA itself undergo LLPS or general condensation due to genomic repression. Current in vitro condensation studies provide valuable insight into the physical properties of condensates but lack the resolution that would allow for complex kinetic studies of multicomponent condensation. Using a supported lipid bilayer platform (SLB) in tandem with objective based total internal reflection microscopy we observe the 2-dimensional lateral movement of macromolecules at a single molecule level.

Protein-PAINT: Superresolution microscopy with signaling proteins.

Superresolution techniques have advanced our understanding of complex cellular structures and processes but require the attachment of fluorophores to targets through tags or antibodies, which can be bulky and result in underlabeling. To overcome these limitations, we developed a technique to visualize the nanoscale binding locations of signaling proteins by taking advantage of their native interaction domains. Here, we demonstrated that pPAINT (protein point accumulation in nanoscale topography) is a new, single-molecule localization microscopy (SMLM) technique and used it to investigate T cell signaling by visualizing the Src homology 2 (SH2) domain, which is common in signaling molecules. When SH2 domain-containing proteins relocate to the plasma membrane, the domains selectively, transiently, and reversibly bind to preferred phosphorylated tyrosine residues on receptors. This transient binding yields the stochastic blinking events necessary for SMLM when observed with total internal reflection microscopy and enables quantification of binding coefficients in intact cells. We used pPAINT to reveal the binding sites of several T cell receptor-proximal signaling molecules, including Zap70, PI3K, Grb2, Syk, Eat2, and SHP2, and showed that the probes could be multiplexed. We showed that the binding half-life of the tandem SH2 domain of PI3K correlated with binding site cluster size at the immunological synapses of T cells, but that longer binding lifetimes were associated with smaller clusters for the monovalent SH2 domain of Eat2. These results demonstrate the potential of pPAINT for investigating phosphotyrosine-mediated signaling processes at the plasma membrane.

Single-molecule investigation of product inhibition of Cel7 cellulase activity by cellobiose

Cel7a is a cellobiohydrolase that degrades cellulose and release the disaccharide cellobiose. To study the product-inhibition mechanism of the Cel7a from Trichoderma reesei, we developed a multimodal microscope and investigated the interaction of Cel7a with immobilized cellulose in the presence of cellobiose. Our custom-built microscope can image immobilized cellulose by interference reflection microscopy and localize binding of quantum dot-labeled Cel7a enzymes by TIRF with a precision of 2.8 nm at 100 frames/s.

Use of sub-cellular fluctuation imaging (SCFI) as a rapid antimicrobial susceptibility test

A rapid Antimicrobial Susceptibility Test (AST) facilitates proper diagnostics and prescriptions, decelerating the progression of antimicrobial resistance. We developed a rapid AST technique called Sub-Cellular Fluctuation Imaging (SCFI) in which bacteria absorbed onto a glass surface are observed using Total Internal Reflection microscopy. Under an evanescent illumination, SCFI detects a fluctuating scattering signal from within a bacterium. To locate the origin of this signal, we measured the SCFI-based fluctuation level at different penetration depths of the evanescent field and found that the relationship is consistent with moving scatterers within the cytoplasm of Escherichia coli (E.

Measurements and simulations of microtubule growth imply strong longitudinal interactions and reveal a role for GDP on the elongating end

In the present study, we used interference reflection microscopy (IRM) to observe the in vitro growth rates and fluctuations of bovine brain microtubules at high temporal resolution. We performed measurements in the presence of GTP and in the presence of the hydrolysis-resistant GTP analog GMPCPP. We fit a computational to the resulting measurements to provide insight into the molecular interactions governing microtubule elongation. Microtubules growing with GMPCPP showed a very low apparent critical concentration, reflecting high affinity interactions between αβ-tubulin subunits and the microtubule end.

A machine learning approach to map crystal orientation by optical microscopy

Mapping grain orientation in crystalline solids is essential to investigate the relationships between local microstructure and crystallography and interpret materials properties. One of the main techniques used to perform these studies is electron backscatter diffraction (EBSD). Due to the limited measurement throughput, however, EBSD is not suitable for characterizing samples with long-range microstructure heterogeneity, nor for building large material libraries that include numerous specimens. We present a machine learning approach for high-throughput crystal orientation mapping, which relies on the optical technique called directional reflectance microscopy. We successfully apply our method on Inconel 718 specimens produced by additive manufacturing, which exhibit complex, spatially-varying microstructures. These results demonstrate that optical orientation mapping on a metal alloy is achievable. Since our method is data-driven, it can be easily extended to different alloy systems produced using different manufacturing processes.

In Vitro Reconstitution of Kinesin-Based, Axonal mRNA Transport.

Motor protein-driven transport of mRNAs on microtubules and their local translation underlie important neuronal functions such as development, growth cone steering, and synaptic plasticity. While there is abundant data on how membrane-bound cargoes such as vesicles, endosomes, or mitochondria are coupled to motor proteins, surprisingly little is known on the direct interactions of RNA-protein complexes and kinesins or dynein. Provided the potential building blocks are identified, in vitro reconstitutions coupled to Total Internal Reflection Microscopy (TIRF-M) are a powerful and highly sensitive tool to understand how single molecules dynamically interact to assemble into functional complexes. Here we describe how we assemble TIRF-M imaging chambers suitable for the imaging of single protein-RNA complexes. We give advice on optimal sample preparation procedures and explain how a minimal axonal mRNA transport complex can be assembled in vitro. As these assays work at picomolar-range concentrations of proteins and RNAs, they allow the investigation of molecules that cannot be obtained at high concentrations, such as many large or disordered proteins. This now opens the possibility to study how RNA-binding proteins(RBPs), RNAs, and microtubule-associated proteins act together in real-time at single-molecule sensitivity to create cytoplasmic mRNA distributions.