Form Of Microscopy Research Articles

Multichannel measurements of C. elegans largest Lyapunov exponents using optical diffraction.

Dynamic diffraction (DOD) is a form of microscopy that allows the dynamic tracking of changing shapes in a 1D time series. DOD can capture the locomotion of a nematode while swimming freely in a 3D space, allowing the locomotion of the worm to more closely mimic natural behavior than in some other laboratory environments. More importantly, we are able to see markers of chaos as DOD covers dynamics on multiple length scales. This work introduces a multichannel method to measure the dynamic complexity of microscopic organisms. We show that parameters associated with chaos, such as the largest Lyapunov exponent (LLE), the mean frequency, mutual information (MI), and the embedding dimension, are independent of the specific point sampled in the diffraction pattern, thus demonstrating experimentally the consistency of our dynamic parameters sampled at various locations (channels) in the associated optical far-field pattern.

Concurrent phase retrieval for imaging strain in nanocrystals

Coherent diffraction imaging is a form of microscopy that permits high resolution imaging of atomic displacements from equilibrium where the use of conventional optics is not feasible. Approaches to date for the recovery of atomic displacements from equilibrium and subsequently strain information occur after phase reconstruction of the complex real-space images from at least three independent Bragg diffraction amplitude measurements. While this is a more accessible and effective approach to recover strain information, there is potential for erroneous results if the recovered phase information is not carefully treated. Here we present a strategy for imaging strain with coherent x rays that eliminates the technical challenges that exist in conventional approaches by constructing the strain field concurrently during the phase retrieval process of recovering phase information.

Observation of diffraction contrast in scanning helium microscopy

Scanning helium microscopy is an emerging form of microscopy using thermal energy neutral helium atoms as the probe particle. The very low energy combined with lack of charge gives the technique great potential for studying delicate systems, and the possibility of several new forms of contrast. To date, neutral helium images have been dominated by topographic contrast, relating to the height and angle of the surface. Here we present data showing contrast resulting from specular reflection and diffraction of helium atoms from an atomic lattice of lithium fluoride. The signature for diffraction is evident by varying the scattering angle and observing sharp features in the scattered distribution. The data indicates the viability of the approach for imaging with diffraction contrast and suggests application to a wide variety of other locally crystalline materials.

Efficacy of USB microscopes for imaging forensic materials

USB microscopy, a relatively new and developing technology, offers a highly portable and relatively inexpensive means for performing microscopy. However, it has not been established that USB microscopes are capable of imaging at a level deemed sufficient for forensic requirements. The purpose of this study was to determine the value, applicability, and limitations of current USB microscope technology by comparing USB microscopes with traditional microscopes by collecting images of common forensic trace materials. For our tests, we considered image clarity, resolution, magnification, field of view, working distance, ease of use, and reflected versus transmission mode imaging. The USB microscopes were highly portable, easy to work with, and able to take an image in any orientation. They also demonstrated a strong sensitivity to different hues of color and were easily tailored to different forms of microscopy by utilizing additional optical components. USB microscopes do not share the same level of resolution, image quality, or image contrast as the most sophisticated microscopes. However, USB microscopes could add value to forensic investigations when used for preliminary exams, low magnification imaging of samples, and imaging scenarios that require magnification but don’t conform to traditional microscopy.

Calvin F. Quate (1923-2019).

Nanoscience pioneer who invented advanced microscopes

ПОРАЖЕНИЕ СВИНИНЫ ТКАНЕВЫМИ ЦИСТАМИ, КОНТРОЛЬ СЫРЬЯ ПРИ САРКОЦИСТОЗЕ

The aim of the study was to evaluate and compare the effectiveness of methods of post-slaughter diagnosis of pig sarcocystosis, analysis of the risk of pork by tissue cysts. The objectives of the study were conduction of veterinary and sanitary examination and diagnosis of pig sarcocystosis using visual inspection and layered dissection of the muscles of the esophagus, pharynx, heart, tongue, diaphragm and carcass muscles, implementation of the compressor method of research and the method of digesting tissues in artificial gastric juice, studying the existing scientific literature on the problems of pig sarcocystosis and its danger. The objects of the study were samples from 70 pigs. The necessity of prevention and careful diagnostics of sarcocystosis of pigs is shown. The efficiency of using additional methods of research in the form of microscopy of sections and the method of artificial tissue digestion is shown.

Using elongated microparticles to enhance tailorable nanoemulsion delivery in excised human skin and volunteers

This study demonstrates, for the first time, clinical testing of elongated silica microparticles (EMP) combined with tailorable nanoemulsions (TNE) to enhance topical delivery of hydrophobic drug surrogates. Likewise, this is the first report of 6-carboxyfluorescein (a model molecule for topically delivered hydrophobic drugs) AM1 & DAMP4 (novel short peptide surfactants) used in volunteers. The EMP penetrates through the epidermis and stop at the dermal-epidermal junction (DEJ). TNE are unusually stable and useful because the oil core allows high drug loading levels and the surface properties can be easily controlled. At first, we chose alginate as a crosslinking agent between EMP and TNE. We initially incorporated a fluorescent lipophilic dye, DiI, as a hydrophobic drug surrogate into TNE for visualization with microscopy. We compared four different coating approaches to combine EMP and TNE and tested these formulations in freshly excised human skin. The delivery profile characterisation was imaged by dye- free coherent anti-Stoke Raman scattering (CARS) microscopy to detect the core droplet of TNE that was packed with pharmaceutical grade lipid (glycerol) instead of DiI. These data show the EMP penetrating to the DEJ followed by controlled release of the TNE. Freeze-dried formulations with crosslinking resulted in a sustained release profile, whereas a freeze-dried formulation without crosslinking showed an immediate burst-type release profile. Finally, we tested the crosslinked TNE coated EMP formulation in volunteers using multiphoton microscopy (MPM) and fluorescence-lifetime imaging microscopy (FLIM) to document the penetration depth characteristics. These forms of microscopy have limitations in terms of image acquisition speed and imaging area coverage but can detect fluorescent drug delivery through the superficial skin in volunteers. 6-Carboxyfluorescein was selected as the fluorescent drug surrogate for the volunteer study based on the similarity of size, charge and hydrophobicity characteristics to small therapeutic drugs that are difficult to deliver through skin. The imaging data showed a 6-carboxyfluorescein signal deep in volunteer skin supporting the hypothesis that EMP can indeed enhance the delivery of TNE in human skin. There were no adverse events recorded at the time of the study or after the study, supporting the use of 6-carboxyfluorescein as a safe and detectable drug surrogate for topical drug research. In conclusion, dry formulations, with controllable release profiles can be obtained with TNE coated EMP that can effectively enhance hydrophobic payload delivery deep into the human epidermis.

Synthetic membrane characterisation – a review: part I

Membrane characterisation provides a crucial link between the preparation and performance of membranes and their structure, chemistry, morphology, transport properties and other characteristics. This feature article – published in two parts – covers various characterisation techniques. The first instalment, which appears here, looks at why it is neccesary to carry out membrane characterisation and provides details of common methods that are used. It describes techniques for studying membrane morphology, such as various forms of microscopy – scanning electron, transmission electron, atomic force, chemical force, positron annihilation lifetime and electron paramagnetic resonance. It also highlights neutron scattering, and wide-angle X-ray and small-angle X-ray scattering.

The Variable Internal Structure of the Mycoplasma penetrans Attachment Organelle Revealed by Biochemical and Microscopic Analyses: Implications for Attachment Organelle Mechanism and Evolution.

Although mycoplasmas have small genomes, many of them, including the HIV-associated opportunist Mycoplasma penetrans, construct a polar attachment organelle (AO) that is used for both adherence to host cells and gliding motility. However, the irregular phylogenetic distribution of similar structures within the mycoplasmas, as well as compositional and ultrastructural differences among these AOs, suggests that AOs have arisen several times through convergent evolution. We investigated the ultrastructure and protein composition of the cytoskeleton-like material of the M. penetrans AO with several forms of microscopy and biochemical analysis, to determine whether the M. penetrans AO was constructed at the molecular level on principles similar to those of other mycoplasmas, such as Mycoplasma pneumoniae and Mycoplasma mobile We found that the M. penetrans AO interior was generally dissimilar from that of other mycoplasmas, in that it exhibited considerable heterogeneity in size and shape, suggesting a gel-like nature. In contrast, several of the 12 potential protein components identified by mass spectrometry of M. penetrans detergent-insoluble proteins shared certain distinctive biochemical characteristics with M. pneumoniae AO proteins, although not with M. mobile proteins. We conclude that convergence between M. penetrans and M. pneumoniae AOs extends to the molecular level, leading to the possibility that the less organized material in both M. pneumoniae and M. penetrans is the substance principally responsible for the organization and function of the AO.IMPORTANCEMycoplasma penetrans is a bacterium that infects HIV-positive patients and may contribute to the progression of AIDS. It attaches to host cells through a structure called an AO, but it is not clear how it builds this structure. Our research is significant not only because it identifies the novel protein components that make up the material within the AO that give it its structure but also because we find that the M. penetrans AO is organized unlike AOs from other mycoplasmas, suggesting that similar structures have evolved multiple times. From this work, we derive some basic principles by which mycoplasmas, and potentially all organisms, build structures at the subcellular level.

Optical breakdown-driven mesostructure in bulk of soda-lime glass

A study of structural changes in soda-lime glass irradiated with a tightly focused nanosecond laser pulse is presented with center on the material area strongly affected by pressure of a shock wave and the induced temperature. Different forms of microscopy, together with photoluminescence and Raman spectroscopy were used in characterization of the structural transitions induced by the optical breakdown in the bulk of soda-lime glass. Inspection of the irradiated region and its vicinity confirmed existence of an elongated void surrounded by a shell of densified material and the outer cracks. Laser-induced material densification was also identified by μ-Raman spectroscopy. High-resolution transmission electron microscopy (HRTEM) of the shock-affected region revealed nano-crystallization. Nanocrystals (crystallites) with an average diameter of 4–5nm were precipitated in a matrix of densified glass and formed in this way a mesoscopic phase embedded in an amorphous host medium. This phase was spatially limited to a layer with a thickness of 100–150nm at the shell/void interface.

Voltammetric Scanning Electrochemical Cell Microscopy: Dynamic Imaging of Hydrazine Electro-oxidation on Platinum Electrodes.

Voltammetric scanning electrochemical cell microscopy (SECCM) incorporates cyclic voltammetry measurements in the SECCM imaging protocol, by recording electrochemical currents in a wide potential window at each pixel in a map. This provides much more information compared to traditional fixed potential imaging. Data can be represented as movies (hundreds of frames) of current (over a surface region) at a series of potentials and are highly revealing of subtle variations in electrode activity. Furthermore, by combining SECCM data with other forms of microscopy, e.g. scanning electron microscopy and electron backscatter diffraction data, it is possible to directly relate the current-voltage characteristics to spatial position and surface structure. In this work we use a "hopping mode", where the SECCM pipet probe is translated toward the surface at a series of positions until meniscus contact. Small amounts of residue left on the surface, upon probe retraction, demark the precise area of each measurement. We use these techniques to study hydrazine oxidation on a polycrystalline platinum substrate both in air and in a deaerated environment. In both cases, the detected faradaic current shows a structural dependence on the surface crystallographic orientation. Significantly, in the presence of oxygen (aerated solution) the electrochemical current decreases strongly for almost all grains (crystallographic orientations). The results highlight the flexibility of voltammetric SECCM for electrochemical imaging and present important implications for hydrazine electroanalysis.

Lightsheet-based fluorescence microscopy (LSFM) for the quantitative imaging of cells and tissues.

In lightsheet-based fluorescence microscopy (LSFM), only the focal plane is illuminated by a laser light sheet. Hence, only the fluorophores within a thin volume of the specimen are excited. This reduces photo-bleaching and photo-toxic effects by several orders of magnitude compared with any other form of microscopy. Therefore, LSFM (aka single/selective-plane illumination microscopy [SPIM] or digitally scanned light sheet microscopy [DSLM]) is the technique of choice for the three-dimensional imaging of live or fixed and of small or large three-dimensional specimens. The parallel recording of millions of pixels with modern cameras provides an extremely fast acquisition speed. Recent developments address the penetration depth, the resolution and the recording speed of LSFM. The impact of LSFM on research areas such as three-dimensional cell cultures, neurosciences, plant biology and developmental biology is increasing at a rapid pace. The development of high-throughput LSFM is the next leap forward, allowing the application of LSFM in toxicology and drug discovery screening.

Lysosome fusion in cultured mammalian cells.

In mammalian cells, lysosomes fuse with late endosomes to form endolysosomes from which lysosomes are reformed. Lysosomal fusion events were initially inferred from light and electron microscopy studies, demonstrated in cell-free content mixing assays and, more recently, shown directly with live cell microscopy. Currently, there is a focus on studying lysosome fusion in cultured cells using various forms of microscopy, especially under conditions in which the use of overexpression of dominant-negative protein constructs or the use of RNA interference to deplete individual proteins allows the investigation of the molecular machinery of fusion. Here, we review a variety of fluorescence, live cell, and electron microscopy techniques with which to study lysosome fusion in cultured mammalian cells. We address the merits and limitations of different techniques when choosing an assay system and provide a series of protocols with which to study endocytic delivery to lysosomes and fusion events between lysosomes and endosomes.

Direct Observation of Unstained Biological Specimens in Water by the Frequency Transmission Electric-Field Method Using SEM

Scanning electron microscopy (SEM) is a powerful tool for the direct visualization of biological specimens at nanometre-scale resolution. However, images of unstained specimens in water using an atmospheric holder exhibit very poor contrast and heavy radiation damage. Here, we present a new form of microscopy, the frequency transmission electric-field (FTE) method using SEM, that offers low radiation damage and high-contrast observation of unstained biological samples in water. The wet biological specimens are enclosed in two silicon nitride (SiN) films. The metal-coated SiN film is irradiated using a focused modulation electron beam (EB) at a low-accelerating voltage. A measurement terminal under the sample holder detects the electric-field frequency signal, which contains structural information relating to the biological specimens. Our results in very little radiation damage to the sample, and the observation image is similar to the transmission image, depending on the sample volume. Our developed method can easily be utilized for the observation of various biological specimens in water.

FibrilTool, an ImageJ plug-in to quantify fibrillar structures in raw microscopy images

Cell biology heavily relies on the behavior of fibrillar structures, such as the cytoskeleton, yet the analysis of their behavior in tissues often remains qualitative. Image analysis tools have been developed to quantify this behavior, but they often involve an image pre-processing stage that may bias the output and/or they require specific software. Here we describe FibrilTool, an ImageJ plug-in based on the concept of nematic tensor, which can provide a quantitative description of the anisotropy of fiber arrays and their average orientation in cells, directly from raw images obtained by any form of microscopy. FibrilTool has been validated on microtubules, actin and cellulose microfibrils, but it may also help analyze other fibrillar structures, such as collagen, or the texture of various materials. The tool is ImageJ-based, and it is therefore freely accessible to the scientific community and does not require specific computational setup. The tool provides the average orientation and anisotropy of fiber arrays in a given region of interest (ROI) in a few seconds.

Laser-induced periodic alignment of Ag nanoparticles in soda-lime glass

One-, two- or three-dimensional arrays of closely spaced silver nanoparticles may lead to new optical properties, due to short or long range coupling between their resonant surface plasmons, so that the spatially controlled growth of silver nanoparticles provides an efficient way to tune their optical properties. Towards this way, we present here the periodic pattern of a glass surface with silver nanoparticles by continuous ultraviolet laser exposure. The formation of the 160 nm period pattern is well described by an interference-based model which agrees with the experimental conclusions, mainly obtained by various forms of microscopy. Statistical approach based on the autocorrelation function gives quantitative description about the quality of the order in the periodic structure and about the nanoparticles averaged diameter (80 nm). We also present the optical extinction spectrum of the Laser Induced Periodic Surface Structure (LIPSS)-containing area of the glass, which unusually shows several bands in the visible range. The period of 160 nm of the periodic structure is short enough to allow coupling between nanoparticles, which makes it a possible candidate for plasmon-based optical applications.

Direct imaging of the near field and dynamics of surface plasmon resonance on gold nanostructures using photoemission electron microscopy

Localized surface plasmon resonance (LSPR) can be supported by metallic nanoparticles and engineered nanostructures. An understanding of the spatially resolved near-field properties and dynamics of LSPR is important, but remains experimentally challenging. We report experimental studies toward this aim using photoemission electron microscopy (PEEM) with high spatial resolution of sub-10 nm. Various engineered gold nanostructure arrays (such as rods, nanodisk-like particles and dimers) are investigated via PEEM using near-infrared (NIR) femtosecond laser pulses as the excitation source. When the LSPR wavelengths overlap the spectrum of the femtosecond pulses, the LSPR is efficiently excited and promotes multiphoton photoemission, which is correlated with the local intensity of the metallic nanoparticles in the near field. Thus, the local field distribution of the LSPR on different Au nanostructures can be directly explored and discussed using the PEEM images. In addition, the dynamics of the LSPR is studied by combining interferometric time-resolved pump-probe technique and PEEM. Detailed information on the oscillation and dephasing of the LSPR field can be obtained. The results identify PEEM as a powerful tool for accessing the near-field mapping and dynamic properties of plasmonic nanostructures. A novel form of microscopy has been used to map the field dynamics of surface plasmon resonances. Hiroaki Misawa and co-workers in Japan used high-resolution (10 nm) multiphoton photoemission electron microscopy (MP-PEEM) to image the field distribution of a variety of metallic nanostructures, such as arrays of gold rods, nanodisks and dimer pairs. MP-PEEM works by recording the electrons emitted from a sample in response to illumination with a series of ultrashort pulses. Illumination with near-infrared pulses caused excited plasmon resonances in the gold nanostructures to show up in the resulting MP-PEEM images as local hot spots, from which the field distribution was determined. The short duration of the pulses made it possible to probe the dynamics of the plasmon resonance field distribution on the femtosecond timescale.

Atomic Force Microscopy: A New Look at Pathogens

Microbial cells are highly complex and heterogeneous systems. In general, cell populations contain subgroups of cells which exhibit differences in growth rate as well as resistance to stress and drug treatment [1]. In addition, individual cells are spatially organized and heterogeneous, and this cellular heterogeneity is used to perform key functions [2]. This complexity emphasizes the need for single-cell analysis techniques in microbial research. Fluorescence imaging is a powerful tool to localize molecules in single cells [3], [4], but the resolution remains limited to the wavelength of the light source. On the other hand, high-resolution images of microbial structures can be obtained by electron microscopy techniques. In particular, cryo-electron tomography—or three-dimensional (3-D) electron microscopy—provides images of whole bacterial cells, at resolutions that are one to two orders of magnitude higher than those obtained with light microscopy [5]. In the past 20 years a new form of microscopy, atomic force microscopy (AFM), has revolutionized the way researchers probe the microbial cell surface. Instead of using an incident beam, AFM measures the minute forces acting between a sharp tip and the sample [6]–[8]. To generate a topographic image, the tip is attached to a cantilever that bends under force and is moved in three-dimensions using a piezoelectric scanner. While scanning the sample surface, cantilever's bending is measured by a laser beam focused on the free end of the cantilever and reflected into a photodiode. Unlike other microscopy techniques, 3-D images of cells and membranes are obtained at high resolution without staining, labelling or fixation, thus in physiological conditions. AFM is much more than a surface-imaging tool in that it also measures the localization and mechanical properties of the individual cell surface molecules. In this modality, known as single-molecule force spectroscopy, the cantilever deflection is recorded as a function of the vertical displacement of the scanner (as the sample is pushed towards the tip and it retracts) [6], [7]. This results in a cantilever deflection vs. scanner displacement curve, which is transformed into a force-distance curve using appropriate corrections. The characteristic adhesion force between tip and sample measured during retraction is used to probe the distribution and mechanics of single molecules, such as cell surface receptors. These novel AFM techniques complement traditional methods used to analyse microbial cell walls and provide new opportunities for understanding cell surface interactions. In this review, we provide a flavour of the various applications offered by AFM in microbiology, and we highlight some of the key breakthroughs the technique has enabled in pathogen research.

Effects of decorin proteoglycan on fibrillogenesis, ultrastructure, and mechanics of type I collagen gels

The proteoglycan decorin is known to affect both the fibrillogenesis and the resulting ultrastructure of in vitro polymerized collagen gels. However, little is known about its effects on mechanical properties. In this study, 3D collagen gels were polymerized into tensile test specimens in the presence of decorin proteoglycan, decorin core protein, or dermatan sulfate (DS). Collagen fibrillogenesis, ultrastructure, and mechanical properties were then quantified using a turbidity assay, 2 forms of microscopy (SEM and confocal), and tensile testing. The presence of decorin proteoglycan or core protein decreased the rate and ultimate turbidity during fibrillogenesis and decreased the number of fibril aggregates (fibers) compared to control gels. The addition of decorin and core protein increased the linear modulus by a factor of 2 compared to controls, while the addition of DS reduced the linear modulus by a factor of 3. Adding decorin after fibrillogenesis had no effect, suggesting that decorin must be present during fibrillogenesis to increase the mechanical properties of the resulting gels. These results show that the inclusion of decorin proteoglycan during fibrillogenesis of type I collagen increases the modulus and tensile strength of resulting collagen gels. The increase in mechanical properties when polymerization occurs in the presence of the decorin proteoglycan is due to a reduction in the aggregation of fibrils into larger order structures such as fibers and fiber bundles.

Photonic crystal enhanced microscopy for imaging of live cell adhesion

A form of microscopy that utilizes a photonic crystal biosensor surface as a substrate for cell attachment enables label-free, quantitative, submicron resolution, time-resolved imaging of cell-surface interactions without cytotoxic staining agents or temporally-unstable fluorophores. Other forms of microscopy do not provide this direct measurement of live cell-surface attachment localization and strength that includes unique, dynamic morphological signatures critical to the investigation of important biological phenomena such as stem cell differentiation, chemotaxis, apoptosis, and metastasis. Here, we introduce Photonic Crystal Enhanced Microscopy (PCEM), and apply it to the study of murine dental stem cells to image the evolution of cell attachment and morphology during chemotaxis and drug-induced apoptosis. PCEM provides rich, dynamic information about the evolution of cell-surface attachment profiles over biologically relevant time-scales. Critically, this method retains the ability to monitor cell behavior with spatial resolution sufficient for observing both attachment footprints of filopodial extensions and intracellular attachment strength gradients.