Use Of Dark-field Microscopy Research Articles

Disruption of endothelial glycocalyx in patients with rheumatoid arthritis

BACKGROUND: Endothelial glycocalyx has an important role in the regulation of endothelial functioning. It is the first to be damaged during the development of autoimmune inflammatory diseases. Thinning of endothelial glycocalyx leads to a violation of the barrier function of blood vessels, the development and maintenance of the inflammatory process. In this context, using endothelial glycocalyx assessment for diagnostic and monitoring of diseases is considered of great interest.
 AIM: To establish the relationship between endothelial glycocalyx thickness with activity, serological profile and risk factors for an unfavorable course of rheumatoid arthritis.
 MATERIALS AND METHODS: The study included 76 patients from 18 to 65 years of both sexes with rheumatoid arthritis. The perfusion boundary region of endothelial glycocalyx determined using a dark-field microscope has been used as the main indicator for assessing the state of endothelial glycocalyx.
 RESULTS: The obtained results have demonstrated a decrease in the initial endothelial glycocalyx thickness in females in the presence of risk factors for an unfavorable course of rheumatoid arthritis. At the same time, no relationship with the serological profile and endothelial glycocalyx thickness has been found. An inverse correlation between endothelial glycocalyx thickness and syndecan-1 level and erythrocyte sedimentation rate has been found. This confirms the fact that endothelial glycocalyxes are involved in the inflammatory process in rheumatoid arthritis. Endothelial glycocalyx assessment using dark-field microscopy made it possible to predict the subsequent appointment of pulse therapy with methylprednisolone in the hospital. On re-examination, endothelial glycocalyx thinning has been observed in the presence of unfavorable risk factors for the course of rheumatoid arthritis, in negative to anti-cyclic citrullinated peptide antibodies patients, or in the absence of response to treatment.
 CONCLUSIONS: The use of dark-field microscopy is unique in its capabilities for intravital and non-invasive assessment of endothelial glycocalyx thickness. It provides important fundamental information deepening knowledge in the pathological physiology of disease. From a clinical point of view, it allows to consider a decrease in endothelial glycocalyx thickness as an additional prognostically unfavorable marker in rheumatoid arthritis.

Luminescent surfaces with tailored angular emission for compact dark-field imaging devices

Dark-field microscopy is a standard imaging technique widely employed in biology that provides high image contrast for a broad range of unstained specimens1. Unlike bright-field microscopy, it accentuates high spatial frequencies and can therefore be used to emphasize and resolve small features. However, the use of dark-field microscopy for reliable analysis of blood cells, bacteria, algae, and other marine organisms often requires specialized, bulky microscope systems, and expensive additional components, such as dark-field-compatible objectives or condensers2,3. Here, we propose to simplify and downsize dark-field microscopy equipment by generating the high-angle illumination cone required for dark field microscopy directly within the sample substrate. We introduce a luminescent photonic substrate with a controlled angular emission profile and demonstrate its ability to generate high-contrast dark-field images of micrometre-sized living organisms using standard optical microscopy equipment. This new type of substrate forms the basis for miniaturized lab-on-chip dark-field imaging devices, compatible with simple and compact light microscopes.

Unstained cell imaging: Morphological insights from coupled fixation and darkfield microscopy

Cell staining techniques are well established in cell biology and associated with a broad range of dedicated dyes; however, they are accompanied by non-negligible costs, preparation time and unavoidable alterations of the sample with foreign molecules. In this context, we point out and propose the use of darkfield microscopy (DM) combined with different fixation protocols (to be used anyway) to enhance the different cell structures and districts as a timesaving and inexpensive support to the techniques that need staining or immuno-staining protocols and products. In a first step, we have analysed the effect of different fixation protocols on DM images for various human cellular lines. The presented imaging study shows that cell morphology actually changes with the fixation protocols that enhance, through contrast and luminosity variations, different shapes and patterns and thus structures of the cells. The different chemical action of various fixations, in fact, modifies the local scattering coefficient, thus affecting in a different way the morphology shown by DM images. As a second step we have compared the observed DM morphologies to those of selective fluorescent staining being therefore able to associate them to specific cell districts (e.g. nucleus, membrane or cytoskeleton).The obtained results indicate that this common microscopy technique can give images with particular cellular structures or districts enhanced more than others depending on the choice of fixation protocol. Therefore Darkfield Microscopy can be considered as a simpler, cheaper and faster method to provide morphological indications, respect to staining techniques, even at low and medium magnifications

Coupled Optical and Electrochemical Probing of Silver Nanoparticle Destruction in a Reaction Layer.

The oxidation of silver nanoparticles is induced to occur near to, but not at, an electrode surface. This reaction at a distance from the electrode is studied through the use of dark‐field microscopy, allowing individual nanoparticles and their reaction with the electrode product to be visualized. The oxidation product diffuses away from the electrode and oxidizes the nanoparticles in a reaction layer, resulting in their destruction. The kinetics of the silver nanoparticle solution‐phase reaction is shown to control the length scale over which the nanoparticles react. In general, the new methodology offers a route by which nanoparticle reactivity can be studied close to an electrode surface.

Comparison of microscopic and molecular enumeration methods for insect viruses: Cryptophlebia leucotreta granulovirus as a case study

Enumeration techniques were compared for quantification of the South African isolate of Cryptophlebia leucotreta granulovirus (CrleGV-SA), used as a biopesticide to control false codling moth (Thaumatotibia leucotreta), an insect pest of various fruits and nuts, including citrus. The routine enumeration method for CrleGV-SA virus particles in experimentation and production of CrleGV-SA biopesticides is dark field microscopy. This method was compared with spectrophotometry, scanning electron microscopy (SEM) and real time quantitative polymerase chain reaction (qPCR). The purpose was to develop an accurate and reliable routine enumeration method for CrleGV-SA occlusion bodies (OBs) and to validate the use of dark field microscopy. Purified and semi-purified CrleGV-SA viral stocks were used. Spectrophotometry was not a suitable or accurate enumeration method. Dark field microscopy and SEM were accurate and statistically comparable (p = 0.064), validating the use of dark field microscopy as an enumeration method for granulovirus (GV). However, SEM has superior resolution and the advantage of easily distinguishing virus particles from debris in semi-purified viral stock preparations. A quantitative PCR technique has been developed based on use of specific oligonucleotide primers for the granulin gene. This has the advantage of not being affected by contamination with non-biological debris or biological material, which impact on the other methods.

Two Anti-radial Spoke Monoclonal Antibodies Inhibit Chlamydomonas Axonemal Motility by Different Mechanisms

In the 9 + 2 axoneme, radial spokes are structural components attached to the A-tubules of the nine outer doublet microtubules. They protrude toward the central pair microtubule complex with which they have transient but regular interactions for the normal flagellar motility to occur. Flagella of Chlamydomonas mutants deficient in entire radial spokes or spoke heads are paralyzed. In this study the importance of two radial spoke proteins in the flagellar movement is exemplified by the potent inhibitory action of two monoclonal antibodies on the axonemal motility of demembranated-reactivated Chlamydomonas models. We show that one of these proteins is localized on the stalk of the radial spokes, whereas the other is a component of the head of the same structure and most likely correspond to radial spoke protein 2 and 1, respectively. Fine motility analysis by videomicrography further indicates that these two anti-radial spoke protein antibodies at low concentration affect motility of demembranated-reactivated Chlamydomonas by changing the flagellar waveform without modifying axonemal beat frequency. They also modify wave amplitude differently during motility inhibition. This brings more direct evidence for the involvement of both radial spoke stalk and head in the fine tuning of the waveform during flagellar motility.

MRNA localization in in vitro grown microtubers of potatoas a tool to study starch metabolism

In vitro grown microtubers were used to investigate whether in situ hybridization is a feasible technique to study expression of genes involved in starch metabolism in potato ( Solanum tuberosum L.). Single node cuttings containing an axillary bud were placed on a tuber-inducing medium. Microtubers were harvested 7 d after culture initiation, and were subsequently prepared for in situ hybridization with antisense RNA probes derived from cDNA clones encoding for sucrose synthase (EC 2.4.1.13), granule bound starch synthase (EC 2.4.1.21) and starch branching enzyme (EC 2.4.1.18). Tissue specific expression can be monitored with the use of dark field microscopy at low magnification. This technique showed that the sucrose synthase gene is expressed in the tuber, but not in the apical meristem or the stolon on which the tuber was formed. Bright field microscopy allows the visualization of mRNAs at the subcellular level. mRNAs encoding for granule bound starch synthase and starch branching enzyme were accumulating primarily around the amyloplast. In contrast, the mRNAs encoding for sucrose synthase did not show a preferential distribution. These results demonstrate the feasibility of in situ hybridization for potato tubers, and suggest an additional level of regulation in starch metabolism.

A New Technique for Visualization of the Human Retinal Vasculature

Adenosine diphosphatase, an endothelial cell ectoenzyme that hydrolyses adenosine 5'-diphosphate to adenosine monophosphate, has been used histochemically to study the human retinal vasculature in flat mounts or as flat-embedded tissue that can be sectioned. In all retinas, reaction product was confined almost exclusively to the vasculature, with arteries having more reaction product than veins or capillaries. Adenosine diphosphatase activity was observed in endothelium, smooth-muscle cells, pericytes, and erythrocytes. In the more elaborate version of the technique, the adenosine diphosphatase-incubated retinas were flat embedded in glycol methacrylate. This allowed study of the retinal vasculature in dual perspective: en bloc with the use of dark-field microscopy and specific areas of interest histologically sectioned for further evaluation. The technique is simple and preserves angioarchitecture because the retinas are intact, and patency of the vasculature can be determined.

Contrast in unstained sections: a comparison of bright and dark field electron microscopy

Resolution in bright field electron micrographs of stained tissue sections is determined primarily by heavy atom clusters required for contrast and visibility of biological structure. Omission of heavy metal stains results in loss of visibility. The use of dark field microscopy on very thin sections is shown to increase the visibility of structure to the degree that heavy atoms can be completely omitted from the preparation. This suggests the use of lighter atom compounds as stains in chemical and cytochemical analyses of tissues, as well as further instrumental developments towards the achievement of higher resolution.

The use of dark-field microscopy for the visualization of acetylcholinesterase in cholinergic neurons.

Acetylcholinesterase (ACEase) was investigated histochemically in hearts, kidneys and urinary bladders of the following animals: man, dog, cat, hamster, rat, tree shrew (Tupaia glis) and lesser bush baby (Galago senegalensis). Slides examined with both bright-field and dark-field illumination showed that use of dark-field microscopy greatly enhanced the visibility of sites of ACEase activity. ACEase-positive nerve fibers which had low enzyme activity were difficult to identify by brght-field, due to their pale staining, but these fibers were easily identified as iridescent structures in the dark-field.

Electron microscope studies of the mechanism of nucleation and growth of Cu Au ii from the disordered state

2014 Thin single crystal films of Cu Au prepared by evaporation have been studied on a hot stage inside the electron microscope. The alloy is disordered by annealing at 450 °C, and the nucleation and growth of ordered regions is studied as the temperature is lowered to some value in the range 380-400 °C. By use of dark field microscopy on superlattice reflections, the distri- bution of ordered nuclei within a disordered matrix is studied in detail, as well as the orientation distribution within the ordered nuclei. This latter distribution is explained in terms of (101) twinning of the Cu Au. Evidence is obtained that dislocations modify the growth of the ordered phase in their vicinity, and that in some circumstances preferential nucleation of order occurs on the dislocations. These observations are being compared with studies of the nucleation of order produced in bulk alloy which is thinned after the heat treatment. LE JOURNAL DE PHYSIQUE ET LE RADIUM TOME 23, OCTOBRE 1962,