Standard Microscopy Techniques Research Articles

Prevalence and risk factors for intestinal parasitic infections in pregnant women residing in three districts of Bogot\xe1, Colombia

BackgroundIntestinal parasitic infections (IPI) lead to significant morbidity and mortality in pediatric and adult populations worldwide. Intestinal parasitism during pregnancy is of interest as it may affect the health of pregnant women and their offspring. This study determined the prevalence of IPI in pregnant women living in substandard conditions in three urban districts of Bogotá, Colombia. Associations between prevalence and sociodemographic factors, housing, and living conditions were also evaluated.MethodsIn a cross-sectional and community-based study, pregnant women were recruited from three districts of Bogotá. A total of 550 participants answered a questionnaire; 331 of these also provided stool samples, with 233 providing one and 98 providing two stool samples. Questionnaire responses were associated with the presence of intestinal parasites, which was determined using a standard combined microscopy technique including direct wet mount and formol–ether concentration. Results were verified by supplementary examination of 48 stool samples by quantitative polymerase chain reaction (qPCR).ResultsAmong pregnant women who lived in selected poor residential areas in Bogotá, the overall prevalence of intestinal parasitism was 41% with 9% polyparasitism. Pathogenic parasites were present in 1.2% of the 331 participants including Giardia lamblia and Ascaris lumbricoides. Higher prevalence was found for parasites with debated pathogenicity, including Blastocystis hominis (25%), Endolimax nana (15%), Entamoeba coli (8%), and Iodamoeba butschlii (2%). Entamoeba histolytica/dispar complex was also detected (1.5%). When comparing a subset of stool samples using the combined microscopy technique and qPCR, the latter detected a higher 58.3% overall IPI prevalence. Higher prevalence of infections by any intestinal parasite was found in participants who had never been dewormed (p = 0.01). Higher but not statistically significant associations were found between any parasite and women living with a partner, and intestinal polyparasitism and being from a minority group and not having a water sink.ConclusionsThis first study of the prevalence of intestinal parasitism in Bogotá focused on pregnant women living in poverty, found a high prevalence of intestinal parasites of debated pathogenicity, and confirmed a low prevalence of pathogenic intestinal parasites. These results highlight the need for educational interventions to disrupt transmission routes for prevalent parasites.

Quantification of cyanobacterial cells via a novel imaging-driven technique with an integrated fluorescence signature

A novel imaging-driven technique with an integrated fluorescence signature to enable automated enumeration of two species of cyanobacteria and an alga of somewhat similar morphology to one of the cyanobacteria is presented to demonstrate proof-of-concept that high accuracy, imaging-based, rapid water quality analysis can be with conventional equipment available in typical water quality laboratories-this is not currently available. The results presented herein demonstrate that the developed method identifies and enumerates cyanobacterial cells at a level equivalent to or better than that achieved using standard manual microscopic enumeration techniques, but in less time, and requiring significantly fewer resources. When compared with indirect measurement methods, the proposed method provides better accuracy at both low and high cell concentrations. It extends the detection range for cell enumeration while maintaining accuracy and increasing enumeration speed. The developed method not only accurately estimates cell concentrations, but it also reliably distinguishes between cells of Anabaena flos-aquae, Microcystis aeruginosa, and Ankistrodesmus in mixed cultures by taking advantage of additional contrast between the target cell and complex background gained under fluorescent light. Thus, the proposed image-driven approach offers promise as a robust and cost-effective tool for identifying and enumerating microscopic cells based on their unique morphological features.

Fabrication of novel surface plasmon resonance induced visible light driven iridium decorated SnO2 nanorods for degradation of organic contaminants

Novel visible light driven iridium (Ir) nanoparticles decorated SnO2 nanorods photocatalyst were synthesized via facile hydrothermal strategy. The effect of iridium loading on the SnO2 phase, morphology and optical properties of the nanocomposites was investigated by standard spectroscopy and microscopy techniques. Under visible light, Ir/SnO2 (3%) nanocomposite displayed much higher photocatalytic activity and decomposed more than 97% of crystal violet and methylene blue dyes after 60 min irradiation. The photocatalytic activity was highly dependent on the loading content of Ir because of the electron acceptor role played by Ir NPs and thus promoting the separation of electron-hole pairs. The enhanced photocatalytic activity could be due to the efficient electrons transfer from the surface plasmon resonance (SPR) effect of metallic Ir to SnO2 nanorods which can, in turn, extend the lifetime of the charge carriers. Based on the experimental results, a possible degradation mechanism was also elucidated using radical scavengers, which indicates that the OH and O2− play a crucial role in photodegradation reaction.

Phase imaging and synthetic aperture super-resolution via total internal reflection microscopy.

Total internal reflection microscopy is mainly used in its fluorescence mode and is the reference technique to image fluorescent proteins in the vicinity of cell membranes. Here, we show that this technique can easily become a phase microscope by simply detecting the coherent signal resulting from the interference between the field scattered by the probed sample and the total internal reflection. Moreover, combining several illumination angles permits generating synthetic aperture reconstructions with improved resolutions compared to standard label-free microscopy techniques.

Characterization of Cell Membrane Permeability In Vitro Part I: Transport Behavior Induced by Single-Pulse Electric Fields.

Most experimental studies of electroporation focus on permeabilization of the outer cell membrane. Some experiments address delivery of ions and molecules into cells that should survive; others focus on efficient killing of the cells with minimal temperature rise. A basic method for quantifying electroporation effectiveness is measuring the membrane’s diffusive permeability. More specifically, comparisons of membrane permeability between electroporation protocols often rely on relative fluorescence measurements, which are not able to be directly connected to theoretical calculations and complicate comparisons between studies. Here we present part I of a 2-part study: a research method for quantitatively determining the membrane diffusive permeability for individual cells using fluorescence microscopy. We determine diffusive permeabilities of cell membranes to propidium for electric field pulses with durations of 1 to 1000 μs and strengths of 170 to 400 kV/m and show that diffusive permeabilities can reach 1.3±0.4×10−8 m/s. This leads to a correlation between increased membrane permeability and eventual propidium uptake. We also identify a subpopulation of cells that exhibit a delayed and significant propidium uptake for relatively small single pulses. Our results provide evidence that cells, especially those that uptake propidium more slowly, can achieve large permeabilities with a single electrical pulse that may be quantitatively measured using standard fluorescence microscopy equipment and techniques.

Antibacterial effect of the laser-generated Se nanocoatings on Staphylococcus aureus and Pseudomonas aeruginosa biofilms

The antibacterial properties of selenium nanoparticles (Se NPs) were successfully demonstrated in vitro for Staphylococcus aureus and Pseudomonas aeruginosa biofilms. The possible mechanisms of antibacterial impact included the emergence of reactive oxygen species, induced by free radicals on the NP surface and accompanied by subsequent oxidative stress, as well as mechanical decomposition of the mitochondrial membrane. Se nanocoatings were deposited on bare and silver-coated silica glass substrates via inkjet printing with concentrated nanoinks, prepared by infrared laser-ablative processing of a solid Se target in a 50%-isopropyl solution. The resulted porous nanofilms with high-percentage surface coverage, consisting of spherical Se NPs and Se nanorods, were characterized by means of standard microscopy techniques (optical, scanning electron, transmission), UV–vis–IR and EDX spectroscopy.

Untargeted Metabolomics Approach in Halophiles: Understanding the Biodeterioration Process of Building Materials.

The aim of the study was to explore the halophile metabolome in building materials using untargeted metabolomics which allows for broad metabolome coverage. For this reason, we used high-performance liquid chromatography interfaced to high-resolution mass spectrometry (HPLC/HRMS). As an alternative to standard microscopy techniques, we introduced pioneering Coherent Anti-stokes Raman Scattering Microscopy (CARS) to non-invasively visualize microbial cells. Brick samples saturated with salt solution (KCl, NaCl (two salinity levels), MgSO4, Mg(NO3)2), were inoculated with the mixture of preselected halophilic microorganisms, i.e., bacteria: Halobacillus styriensis, Halobacillus naozhouensis, Halobacillus hunanensis, Staphylococcus succinus, Marinococcus halophilus, Virgibacillus halodenitryficans, and yeast: Sterigmatomyces halophilus and stored at 28°C and 80% relative humidity for a year. Metabolites were extracted directly from the brick samples and measured via HPLC/HRMS in both positive and negative ion modes. Overall, untargeted metabolomics allowed for discovering the interactions of halophilic microorganisms with buildings materials which together with CARS microscopy enabled us to elucidate the biodeterioration process caused by halophiles. We observed that halophile metabolome was differently affected by different salt solutions. Furthermore, we found indications for haloadaptive strategies and degradation of brick samples due to microbial pigment production as a salt stress response. Finally, we detected changes in lipid content related to changes in the structure of phospholipid bilayers and membrane fluidity.

The building stone of the Roman city of Dougga (Tunisia): Provenance, petrophysical characterisation and durability

Preservation of the architectural integrity of archaeological sites requires detailed information about the properties of the building materials and their decay processes. This study investigates the petrophysical properties of the Eocene nummulitic limestone rock used in the construction of the Roman City of Dougga (Tunisia) and its resistance to various factors of decay. The petrographical study is carried out using standard microscope techniques and the petrophysical behaviour by a combination of techniques focusing on hydric and mechanical properties. The pore network is studied by fluorescence microscopy and mercury intrusion porosimetry. Durability was assessed by ice crystallization and SO2 attack ageing tests. The results allowed the identification of four main building lithotypes and their exact stratigraphic point of mining in the ancient quarries. The petrographic variations between lithotypes correlate well with their physical properties. In addition, the four lithotypes show high resistance to ageing tests, confirming the excellent characteristics of the studied nummulitic limestones as a building rock.

Suppressed Umklapp scattering of β-FeSi2 thin film and single crystalline nanowires

Thermal conductivity is an intriguing physical property in the nanometer or quantum regime. In this study, we report the growth and thermal conductivity of β-FeSi2 thin film and single crystalline nanowires. The sample structures and chemical composition were identified by standard microscopy techniques. The temperature-dependent thermal conductivity of the thin film was measured by the 3ω method, whereas that of the nanowires was conducted by a suspended pattern technique. Temperature-dependent thermal conductivity was found to increase gradually from 300 to 500 K due to the effect of surface scattering. The experimental evidence presented herein is the suppression of Umklapp transport at the nanoscale near room temperature and the potential application of nanoengineered β-FeSi2 for future design of thermoelectric materials.

Diversity, abundance, and host relationships of avian malaria and related haemosporidians in New Mexico pine forests.

Avian malaria and related haemosporidian parasites (genera Haemoproteus, Plasmodium, and Leucocytozoon) affect bird demography, species range limits, and community structure, yet they remain unsurveyed in most bird communities and populations. We conducted a community-level survey of these vector-transmitted parasites in New Mexico, USA, to describe their diversity, abundance, and host associations. We focused on the breeding-bird community in the transition zone between piñon-juniper woodland and ponderosa pine forests (elevational range: 2,150–2,460 m). We screened 186 birds representing 49 species using both standard PCR and microscopy techniques to detect infections of all three avian haemosporidian genera. We detected infections in 68 out of 186 birds (36.6%), the highest proportion of which were infected with Haemoproteus (20.9%), followed by Leucocytozoon (13.4%), then Plasmodium (8.0%). We sequenced mtDNA for 77 infections representing 43 haplotypes (25 Haemoproteus, 12 Leucocytozoon, 6 Plasmodium). When compared to all previously known haplotypes in the MalAvi and GenBank databases, 63% (27) of the haplotypes we recovered were novel. We found evidence for host specificity at the avian clade and species level, but this specificity was variable among parasite genera, in that Haemoproteus and Leucocytozoon were each restricted to three avian groups (out of six), while Plasmodium occurred in all groups except non-passerines. We found striking variation in infection rate among host species, with nearly universal infection among vireos and no infection among nuthatches. Using rarefaction and extrapolation, we estimated the total avian haemosporidian diversity to be 70 haplotypes (95% CI [43–98]); thus, we may have already sampled ∼60% of the diversity of avian haemosporidians in New Mexico pine forests. It is possible that future studies will find higher diversity in microhabitats or host species that are under-sampled or unsampled in the present study. Fortunately, this study is fully extendable via voucher specimens, frozen tissues, blood smears, parasite images, and documentation provided in open-access databases (MalAvi, GenBank, and ARCTOS).

In situ monitoring of dislocation proliferation during plastic deformation using ultrasound

Ultrasound has long been used as a non-destructive tool to test for the brittle fracture of materials. Could it be used as a similar tool to test for ductile failure? As a first step towards answering this question, we report results of local measurements of the speed of transverse waves in aluminum under standard testing conditions at two different probe locations and continuously as a function of applied load. The result, as expected, is independent of stress in the elastic regime, but there is a clear change, consistent with a proliferation of dislocations, as soon as the yield strength is reached. We use a model that blames the change in wave speed on the interaction of elastic waves with oscillating dislocation segments, which quantitatively relates the change in wave velocity with dislocation density Λ and segment length L, thus obtaining a continuous relation between dislocation density and externally applied stress. We took off samples from the probe before, at intermediate, and high loading, and we measured their dislocation density using standard X-ray diffraction and transmission electron microscopy techniques. The results agree well with the acoustic measurements, and the relation between stress and dislocation density is consistent with the Taylor rule. This indicates that monitoring the speed of transverse waves could become a useful diagnostic of dislocation density for metallic pieces in service as well as a tool to test models of plastic behavior.

Modular low-light microscope for imaging cellular bioluminescence and radioluminescence.

Low-light microscopy methods are receiving increased attention as new applications have emerged. One such application is to allow longitudinal imaging of light-sensitive cells with no phototoxicity and no photobleaching of fluorescent biomarkers. Another application is for imaging signals that are inherently dim and undetectable using standard microscopy techniques, such as bioluminescence, chemiluminescence or radioluminescence. In this protocol, we provide instructions on how to build a modular low-light microscope (1-4 d) by coupling two microscope objective lenses, back to back from each other, using standard optomechanical components. We also provide directions on how to image dim signals such as those of radioluminescence (1-1.5 h), bioluminescence (∼30 min) and low-excitation fluorescence (∼15 min). In particular, radioluminescence microscopy is explained in detail, as it is a newly developed technique that enables the study of small-molecule transport (e.g., radiolabeled drugs, metabolic precursors and nuclear medicine contrast agents) by single cells without perturbing endogenous biochemical processes. In this imaging technique, a scintillator crystal (e.g., CdWO4) is placed in close proximity to the radiolabeled cells, where it converts the radioactive decays into optical flashes detectable using a sensitive camera. Using the image reconstruction toolkit provided in this protocol, the flashes can be reconstructed to yield high-resolution images of the radiotracer distribution. With appropriate timing, the three aforementioned imaging modalities may be performed together on a population of live cells, allowing the user to perform parallel functional studies of cell heterogeneity at the single-cell level.

Brain invasion in meningiomas-clinical considerations and impact of neuropathological evaluation: a systematic review.

With the release of the 2016 edition of the World Health Organization (WHO) Classification of Central Nervous System Tumors, brain invasion in meningiomas has been added as a stand-alone criterion for atypia and can therefore impact grading and indirectly adjuvant therapy. Regarding this rising clinical importance, we have reviewed the current knowledge about brain invasion with emphasis on its implications on current and future clinical practice. We found various definitions of brain invasion and approaches for evaluation in surgically obtained specimens described over the past decades. This heterogeneity is reflected by weak correlation with prognosis and remains controversial. Similarly, associated clinical factors are largely unknown. Preoperative, imaging-guided detection of brain invasion is unspecific, and intraoperative assessment using standard and new high-magnification microscopic techniques remains imprecise. Despite the increasing knowledge about molecular alterations of the tumor/ brain surface, pharmacotherapeutic options targeting brain invasive meningiomas are lacking. Finally, we summarize the impact of brain invasion on histopathological grading in the WHO classifications of brain tumors since 1979.In conclusion, standardized neurosurgical sampling and neuropathological analyses could improve diagnostic reliability and reproducibility of future studies. Further research is needed to improve pre- and intraoperative visualization of brain invasion and to develop adjuvant, targeted therapies.

Description of the Sperm and Spermatheca of Hypothenemus hampei (Coleoptera: Curculionidae: Scolytinae) for the Differentiation of Mated and Unmated Females

The coffee berry borer, Hypothenemus hampei (Ferrari), is a major insect pest of coffee crops worldwide. As part of a program of studies aimed at efficient and sustainable pest control, a study was conducted to determine the mating status of adult females in the broader context of studying pest dispersal behavior. To that end, it was deemed important to determine the morphology of the H. hampei female reproductive tract. This study focused on direct observation of the spermatheca of unmated, and colonizing adult females obtained from natural field populations with a high probability of having mated, and the description of all organs of the reproductive tract using established standard staining and microscopy techniques. Spermathecae were extracted from the samples and sorted according to their origin (unmated or colonizing females). The morphology of sperm present in the female spermathecae was compared with that of sperm taken directly from male testes. Two staining techniques using Giemsa and chlorazol black allowed spermatheca and sperm to be distinguished from other tissues. Under these conditions, a more precise description of the spermatheca was achieved, including the spermathecal duct and spermathecal muscles, spermathecal gland, and, for the first time, sperm when present. These techniques permitted the accurate and rapid determination of the presence or absence of sperm in the spermathecae of H. hampei females, thereby establishing their mating status. (Resume d'auteur)

Outcomes of Using Otoendoscopy During Surgery for Cholesteatoma.

To determine the impact of using otoendoscopy at the time of primary surgery of cholesteatoma in identifying hidden "cholesteatoma remnant". Study was prospective study. Setting was University tertiary care hospital. One hundred fifty, patients diagnosed clinically and by CT as having cholesteatoma, have been operated. 64 patients operated by using canal up technique and 86 patients operated by using canal down technique. Once all visible cholesteatoma was removed with standard microscopic techniques, otoendoscopy was utilized in every patient to identify any hidden "cholesteatoma remnant". Despite apparent total microscopic eradication of cholesteatoma of the operated cases, otoendoscopy at time of primary surgery revealed an overall incidence of hidden cholesteatoma remnants of 18%. The incidence of hidden cholesteatoma remnants identified by otoendoscopy was 23% in the canal up group and 14% in the canal down group. Otoendoscopy should be used as an adjunct with standard microscopic technique to identify hidden cholesteatoma remnants during surgery of cholesteatoma.

A simple 2D composite image analysis technique for the crystal growth study of L-ascorbic acid.

This work was destined for 2D crystal growth studies of L-ascorbic acid using the composite image analysis technique. Growth experiments on the L-ascorbic acid crystals were carried out by standard (optical) microscopy, laser diffraction analysis, and composite image analysis. For image analysis, the growth of L-ascorbic acid crystals was captured as digital 2D RGB images, which were then processed to composite images. After processing, the crystal boundaries emerged as white lines against the black (cancelled) background. The crystal boundaries were well differentiated by peaks in the intensity graphs generated for the composite images. The lengths of crystal boundaries measured from the intensity graphs of composite images were in good agreement (correlation coefficient "r" = 0.99) with the lengths measured by standard microscopy. On the contrary, the lengths measured by laser diffraction were poorly correlated with both techniques. Therefore, the composite image analysis can replace the standard microscopy technique for the crystal growth studies of L-ascorbic acid.

Artifacts in histology: A 1-year retrospective study

Introduction: Histology is a science of the analysis of tissue architecture; however, the presence of artifacts in microscopic sections may result in misdiagnosis. Despite documented common occurrences, studies on the patterns of artifacts in Nigeria are however scant. The rarity of descriptions in this clumsy but important component of histology stimulated our interest in demonstrating the various patterns of artifacts in a laboratory. This 1-year retrospective study was conducted in Federal Medical Centre, Asaba, Delta State. Materials and Methods: Tissue sections were viewed and with the aid of a microscope to check for the various patterns of artifacts. These artifacts were seen as artificial structures or tissue alternations on the prepared slide. Histological images were captured using eyepiece Scopetek DCM 500, 5.0 Megapixel connected USB 2.0 computer. Data were obtained by standard microscopic techniques in which the various patterns of tissue alterations were described. Permission for this study was obtained from the hospital Ethics Committee (ethical number FMC/ASB/T/A81/198). Results: This review of patterns of artifacts showed that during the 1-year period, of the 388 slides reviewed, 94.59% had the presence of artifacts. The results also revealed that fold artifacts were the most prevalent patterns constituting about 33.00% of the total tissue sections observed, followed by artifacts attributed to microtomy which accounted for 18.47% and formalin pigment artifacts, 14.78%. The least was heat and hemorrhagic artifacts which contributed to about 0.25%. Conclusion: In conclusion, fold artifacts were the most prevalent patterns observed in this study due to the thin sections which easily stretch around other structures having different constituencies if the tissue is not carefully lifted from the water bath.

Analysis of Coronary Vessels in Cleared Embryonic Hearts

Whole mount visualization of the embryonic coronary plexus from which the capillary and arterial networks will form is rendered problematic using standard microscopy techniques, due to the scattering of imaging light by the thick heart tissue, as these vessels are localized deep within the walls of the developing heart. As optical clearing of tissues using organic solvents such as BABB (1 part benzyl alcohol to 2 parts benzyl benzoate) has been shown to greatly improve the optical penetration depth that can be achieved, we combined clearance of whole, PECAM1-immunostained hearts, with laser-scanning confocal microscopy, in order to obtain high-resolution images of vessels throughout the entire heart. BABB clearance of embryonic hearts takes place rapidly and also acts to preserve the fluorescent signal for several weeks; in addition, samples can be imaged multiple times without loss of signal. This straightforward method is also applicable to imaging other types of blood vessels in whole embryos.

Analysis of Coronary Vessels in Cleared Embryonic Hearts.

Whole mount visualization of the embryonic coronary plexus from which the capillary and arterial networks will form is rendered problematic using standard microscopy techniques, due to the scattering of imaging light by the thick heart tissue, as these vessels are localized deep within the walls of the developing heart. As optical clearing of tissues using organic solvents such as BABB (1 part benzyl alcohol to 2 parts benzyl benzoate) has been shown to greatly improve the optical penetration depth that can be achieved, we combined clearance of whole, PECAM1-immunostained hearts, with laser-scanning confocal microscopy, in order to obtain high-resolution images of vessels throughout the entire heart. BABB clearance of embryonic hearts takes place rapidly and also acts to preserve the fluorescent signal for several weeks; in addition, samples can be imaged multiple times without loss of signal. This straightforward method is also applicable to imaging other types of blood vessels in whole embryos.

Optical photon reassignment with increased axial resolution by structured illumination

Fluorescent microscopy methods linked to the reassignment principle as image scanning microscopy (ISM), re-scan confocal (RSC), optical photon reassignment (OPRA) and instant structured illumination microscopy (iSIM) have the potential to replace confocal microscopy as the standard microscopy technique. Photon reassignment methods are known to link the most important properties in biological imaging as resolution, sensitivity, imaging speed and combinability with fluorophores in an elegant way. On the example of OPRA, we show how this method could be easily extended to the third dimension. If OPRA is used in combination with a structured illumination pattern the sectioning ability can be improved while maintaining the very high signal intensity. We present a detailed analysis about the imaging properties of OPRA in three dimensions and show experimental results on biological samples.