Differential Interference Contrast Optics Research Articles

Focal Recording of Synaptic Currents from Single Boutons at the Drosophila Larval Neuromuscular Junction.

Focal recording is an extracellular method for studying synaptic transmission at the Drosophila larval neuromuscular junction (NMJ) designed for the study of synaptic activity of one or a few synaptic boutons rather than the ensemble activity of all the boutons as occurs with intracellular recording or two-electrode voltage-clamp. This is a useful technique for investigating the properties of different motor neurons that innervate the same muscle, applying statistical analysis to discrete synaptic events, and investigating the heterogeneity of synaptic release properties among boutons. A compound microscope with epifluorescent imaging capability is very helpful but not essential; any GFP Drosophila strain that labels the nerve terminal or synaptic boutons can be used to locate the boutons. A particularly useful strain is Mhc-CD8-Sh-GFP, containing a GFP molecule that is expressed in muscle, localizes to the postsynaptic apparatus, and outlines boutons. Vital fluorescent dyes (such as 4-Di-2-Asp) may also be applied to the dissected preparation to help locate boutons. The microscope should be equipped for differential interference contrast (DIC or Nomarski) optics if fluorescence is not used.

Morphological and Molecular Characterization of Two New Species of Ceratomyxa Thélohan, 1892 (Cnidaria: Myxosporea) from the Marine Ornamental Fish Zanclus cornutus (Linnaeus, 1758) off Lakshadweep Islands, Arabian Sea.

The present study provides the complete morphological and molecular description of two new species of myxosporeans, Ceratomyxa zancli n. sp. and Ceratomyxa cornuti n. sp. infecting the gallbladder of Zanclus cornutus from the Lakshadweep Islands, Arabian Sea. Zanclus cornutus were screened for the presence of myxosporeans, and the recovered myxospores were morphologically characterized using Nomarski Differential Interference Contrast (DIC) optics. The sequences of SSU rDNA were employed for molecular and phylogenetic studies. Both the parasites exhibited a prevalence of 21% each. C. zancli n. sp. is characterized by broadly cresentic myxospores with convex anterior and slightly concave to straight posterior margins and rounded ends. Spore valves two, unequal, measured 9.6 ± 0.7μm × 25.2 ± 1.3μm. Polar capsules two, unequal, spherical, measured 4 ± 0.6μm × 3.5 ± 0.6μm. Polar filament exceptionally long and arranged irregularly. Myxospores of C. cornuti n. sp. are elongated with convex anterior and slightly concave to straight posterior margins. Spore valves two, unequal, measured 7.00 ± 0.4μm × 26.56 ± 1.8μm. Polar capsules spherical, unequal, measured 3.52 ± 0.2 × 3.36 ± 0.35. Molecular analysis of C. zancli n. sp. (ON818297) and C. cornuti n. sp. (ON818298) resulted in 1469 and 1491bp long SSU rDNA sequences, respectively. Molecularly C. zancli n. sp. is close to C. diplodae and C. barnesi with 91.39% similarity, while C. cornuti n. sp. appears closer to C. robertsthomsoni with 97.46% similarity. In phylogenetic analyses, C. zancli n. sp. branched separately within the Ceratomyxa clade while C. cornuti n. sp. clustered with C. robertsthomsoni and C. thalassomae. Based on the differences in morphological, morphometric, molecular, and phylogenetic characteristics, as well as differences in the host and geographic location, the above two species of myxosporeans are considered novel. The study forms the first report of a species of Ceratomyxa from Z. cornutus.

In vitro laser scanning confocal microscopy and unsupervised segmentation: Quantification of cytosolic calcium and RNA distribution in hypoxic neurons.

The mimicry of neurodegenerative diseases in vitro can be observed through the induction of chronic hypoxia, and the impact of this stress is monitored using multiplexed imaging techniques. While laser scanning confocal microscopy (LSCM) is a valuable tool for observing single neurons under degenerative conditions, accurately quantifying RNA distribution and cell size by deep learning tools remains challenging due to the lack of annotated training datasets. To address this, we propose a framework that combines 3D tracking of RNA distribution and cell size identification using unsupervised image segmentation. Additionally, we quantified the calcium level in neurons using fluorescent microscopy using unsupervised image segmentation. First, we performed imaging of neuronal morphology using differential interference contrast (DIC) optics and RNA/calcium level imaging using fluorescent microscopy. Next, we performed k-means clustering-based cell segmentation. The results show that our framework can distinguish between distinct neuronal states under control and chronic hypoxic conditions. The analysis reveals that hypoxia induces a significant increase in cytosolic calcium level, reduction in neuron diameter, and alterations in RNA distribution.Clinical Relevance- The proposed framework is crucial to study the neurodegeneration process and evaluating the efficacy of neuroprotective drugs through image analysis.

Ventral Nerve Cord Dissection and Microscopy of Drosophila Embryos.

The technique of visualizing axon pathways in the embryonic ventral nerve cord using antibody labeling has been fundamental to our understanding of the genetic and developmental mechanisms underlying nervous system wiring in Drosophila. High-resolution microscopic examination of the ventral nerve cord remains an essential component of many experiments in Drosophila developmental neuroscience. Although it is possible to examine the ventral nerve cord in intact whole-mount embryos, to collect the highest-quality images it is often useful to isolate the nervous system away from the other embryonic tissues through embryo dissection. This protocol describes methods for dissecting ventral nerve cords from Drosophila embryos that have been fixed and stained via immunofluorescence or horseradish peroxidase (HRP) immunohistochemistry. The process of making fine dissection needles for this purpose from electrolytically sharpened tungsten wire is also described. Dissected and mounted ventral nerve cords can be examined and imaged using a variety of microscopy techniques including differential interference contrast (DIC) optics, epifluorescence, or confocal microscopy.

Morphological and Molecular Characterization of Myxobolus planilizae n. sp. (Cnidaria; Myxosporea; Myxobolidae) Infecting the Largescale Mullet Planiliza macrolepis (Smith, 1846) Collected From Cochin Backwaters, India.

Myxobolus planilizae n. sp. is described from the intestinal muscles of the largescale mullet Planiliza macrolepis from Cochin backwaters, Kerala, India. Host fishes inhabiting Cochin backwaters were collected using Chinese nets/gill nets. The morphometry and morphological studies were carried out using Nomarski differential interference contrast (DIC) optics, followed by molecular and phylogenetic analyses of the small subunit ribosomal DNA gene (SSU rDNA). Plasmodia small, pale white, and infect the muscles of the intestine; measured 0.13-0.22 (0.17) × 0.09-0.14 (0.13) mm. Mature myxospores pyriform in valvular view, and biconvex in sutural and apical views with a short anterior extension, and measured 7.45-8.75 (8.40) × 6.04-6.86 (6.25) µm. Shell valves with sutural ornamentations. Polar capsules two, equal, pyriform, measured 3.96-4.54 (4.45) × 2.22-2.94 (2.52) µm. Polar filament arranged in five coils, measured 24.41-34.44 (28.52) µm when extruded. In morphological and morphometric analysis, the present species exhibit remarkable variations from other species of the genus Myxobolus. In molecular analysis, the present species revealed the highest identity of 91.85% and divergence of 9.95% with related species, underlining its molecular uniqueness. In phylogenetic analysis, species of Myxobolus infecting mullets appeared as a separate clade and the present species was positioned distinctly with a high bootstrap value. Based on morphology, morphometry, and molecular and phylogenetic analyses, along with tissue/host specificities and geographic location, the present parasite is treated as new and is reported here as M. planilizae n. sp.

Sphaeromyxa cornuti n. sp., a New Species of Myxosporean Infecting the Gallbladder of the Moorish Idol, Zanclus cornutus (Linnaeus, 1758) from Lakshadweep Waters.

The present study describes a new species of myxosporean, Sphaeromyxa cornuti n. sp. infecting the gallbladder of the Moorish idol, Zanclus cornutus (Linnaeus 1758) collected from Lakshadweep waters of the Arabian Sea. Fish were collected using traps and cages. The morphology of mature spores recovered from the gallbladder of Z. cornutus was studied under Nomarski Differential Interference Contrast (DIC) optics. The molecular and phylogenetic analyses were based on SSU rDNA. Sphaeromyxa cornuti n. sp. is characterized by arcuate myxospores with tapering extremities and round ends in valvular, and slightly sigmoid in sutural views (19.2-24.7 µm × 4.1-5.7µm). The two polar capsules are unequally elongate-ovoid in shape and positioned at opposite ends of the spore (6.2-9.7µm × 1.7-2.6µm). Each encloses an irregularly folded, ribbon-like polar tubule, which is oriented parallel to polar capsule axis. In molecular and phylogenetic analyses, the present myxosporean revealed significant differences with related forms and clustered together with S. hellandi within the 'incurvata' group of the Sphaeromyxa clade with high nodal support. Morphological, morphometric, molecular and phylogenetic differences between our material and previously described species of Sphaeromyxa, along with host and geographic variations indicate that the present myxosporean is unique and the name Sphaeromyxa cornuti n. sp. is proposed. This forms the first report of a myxosporean parasite-infecting Z. cornutus.

Iron Repletion Increases Platelet Aggregation and Reduces Platelet Count in Iron Deficient Premenopausal Women

Background: Iron deficiency, heavy menstrual bleeding, and anemia are interrelated conditions with high prevalence in women's health. Together, they impact an estimated 310 million premenopausal women worldwide. Iron deficiency has been associated with development of anemia as well as thrombocytosis. However, the effects of iron deficiency on platelet function remain unknown.Objective: We set out to investigate the impact of IV iron repletion on platelet function, platelet count, and other blood cell indices in iron deficient premenopausal women.Methods: We conducted a prospective single-center study of iron deficient premenopausal women who underwent iron repletion with a single dose of 1000 mg of low molecular weight iron. Pre-infusion and post-infusion blood samples were obtained for laboratory analysis. Standard of care monitoring of iron indices and complete blood counts were also included in the analysis. Pre-infusion rates of anemia and thrombocytosis were calculated using minimum hemoglobin and peak platelet count in the 6 months preceding iron infusion. Platelet function was quantified by Fluorescence-Activated Cell Sorting (FACS) quantification of platelet activation marker antibodies for GPIIb/IIIa (PAC1) and P-selectin (CD62P) after exposure to multiple platelet agonists. Platelet aggregation was assessed by flow of anticoagulated whole blood at a shear rate of 300 s -1 for 5 minutes through chambers coated with type I collagen, imaged with differential interference contrast optics, and quantitatively represented through computation of percent total surface coverage. Pre-infusion and post-infusion cell indices and aggregation data were compared by paired samples t-test. All statistical analyses were performed in GraphPad Prism (Version 8.0.0).Results: Thirteen patients were included in the analysis. Heavy menstrual bleeding was the suspected cause of iron deficiency in 83% of patients. Thrombocytosis was present in 15% of patients at pre-infusion and no patients at post-infusion. Average ferritin was 14 µg/L at pre-infusion and 126 µg/L at post-infusion. The peak platelet count within 6 months pre-infusion was 309 K/mm 3 (±89) vs. 274 (±64) K/mm 3 at post-infusion (p<0.05). The average change to 6-month pre-infusion peak and post-infusion platelet count was -35.2 K/mm 3 (95%CI: -66.2, -5.23). The mean 6-month minimum hemoglobin was 11.9 (±1.9) g/dL vs. 13.3 (±1.1) g/dL at post-infusion (p<0.005). Acquisition of FACS platelet reactivity data are ongoing, with preliminary results for the first 7 consecutively enrolled study patients displayed in Figure 1. Platelet aggregation measured at pre-infusion showed 14% surface coverage, with a significant increase to 29% at post-infusion (p < 0.05). None of the 13 women experienced a thrombotic event during the study period.Conclusion: Correction of iron deficiency results in decreased platelet count and improves platelet aggregation over collagen. Iron repletion may also improve platelet reactivity in response to physiologic agonists. These findings may suggest that iron deficiency impairs hemostasis and that correction of low iron may be even more critical for women with heavy menstrual bleeding. [Display omitted] DisclosuresShatzel: Aronora Inc,: Consultancy.

3D and 4D Tumorigenesis Model for the Quantitative Analysis of Cancer Cell Behavior and Screening for Anticancer Drugs.

Cancer cells from cell lines and tumor biopsy tissue undergo aggregation and aggregate coalescence when dispersed in a 3D Matrigel™ matrix. Coalescence is a dynamic process mediated by a subset of cells within the population of cancer cells. In contrast, non-tumorigenic cells from normal cell lines and normal tissues do not aggregate or coalesce, nor do they possess the motile cell types that orchestrate coalescence of cancer cells. Therefore, coalescence is a cancer cell-specific phenotype that may drive tumor growth in vivo, especially in cases of field cancerization. Here, we describe a simple 3D tumorigenesis model that takes advantage of the coalescence capabilities of cancer cells and uses this feature as the basis for a screen for treatments that inhibit tumorigenesis. The screen is especially useful in testing monoclonal antibodies that target cell-cell interactions, cell-matrix interactions, cell adhesion molecules, cell surface receptors, and general cell surface markers. The model can also be used for 2D imaging in a 96-well plate for rapid screening and is adaptable for 3D high-resolution assessment. In the latter case, we show how the 3D model can be optically sectioned with differential interference contrast (DIC) optics, then reconstructed in 4D and quantitatively analyzed by computer-assisted methods, or, alternatively, imaged with confocal microscopy for 4D quantitative analysis of cancer cell interactions with normal cells within the tumor microenvironment. We demonstrate reconstructions and quantitative analyses using the advanced image analysis software J3D-DIAS 4.2, in order to illustrate the types of detailed phenotypic characterizations that have proven useful. Other software packages may be able to perform similar types of analyses.

Harmonically decoupled gradient light interference microscopy (HD-GLIM).

Differential phase sensitive methods, such as Nomarski microscopy, play an important role in quantitative phase imaging due to their compatibility with partially coherent illumination and excellent optical sectioning ability. In this Letter, we propose a new system, to the best of our knowledge, to retrieve differential phase information from transparent samples. It is based on a 4f optical system with an amplitude-type spatial light modulator (SLM), which removes the need for traditional differential interference contrast (DIC) optics and specialized phase-only SLMs. We demonstrate the principle of harmonically decoupled gradient light interference microscopy using standard samples, as well as static and dynamic biospecimens.

Intracytoplasmic morphologically selected sperm injection, but for whom?

Intracytoplasmic sperm injection (ICSI) is performed in cases of infertility by injecting a motile and morphologically normal sperm cell under a routine ×400 magnification at which is hard to distinguish morphologically healthy sperm. Recently, the use of high-powered differential interference contrast optics gave the opportunity to select a sperm under ultra-high magnification of ×10,160. The aim of the present study was to evaluate the efficacy of the intracytoplasmic morphologically selected sperm injection (IMSI) technique in different infertility populations undergoing ICSI. Main outcome measures of routine ICSI were compared with IMSI in three different groups of patients (1, non-selected; 2, male infertility; and 3, repeated implantation failure group). Results were analysed to evaluate the effects of the IMSI procedure and to find the most suitable group of patients who may benefit from the procedure. IMSI caused a significant increase in the fertilization and top quality embryo rates in the male infertility group and a significant increase in fertilization and pregnancy rates in the repeated implantation failure group, whereas no effect was observed in the non-selected group with patients of various indications. A positive effect of IMSI on the outcome of male factor infertility and repeated implantation failure patients was observed. Data observed confirmed that the application of IMSI was beneficial for a selected group of patients with male factor infertility and repeated implantation failure.

Green Fluorescent Protein Transformation Sheds More Light on a Widespread Mycoparasitic Interaction.

Powdery mildews, ubiquitous obligate biotrophic plant pathogens, are often attacked in the field by mycoparasitic fungi belonging to the genus Ampelomyces. Some Ampelomyces strains are commercialized biocontrol agents of crop pathogenic powdery mildews. Using Agrobacterium tumefaciens-mediated transformation (ATMT), we produced stable Ampelomyces transformants that constitutively expressed green fluorescent protein (GFP) to (i) improve the visualization of the mildew-Ampelomyces interaction and (ii) decipher the environmental fate of Ampelomyces fungi before and after acting as a mycoparasite. Detection of Ampelomyces structures, and especially hyphae, was greatly enhanced when diverse powdery mildew, leaf, and soil samples containing GFP transformants were examined with fluorescence microscopy compared with brightfield and differential interference contrast optics. We showed for the first time, to our knowledge, that Ampelomyces strains can persist up to 21 days on mildew-free host plant surfaces, where they can attack powdery mildew structures as soon as these appear after this period. As saprobes in decomposing, powdery mildew-infected leaves on the ground and also in autoclaved soil, Ampelomyces strains developed new hyphae but did not sporulate. These results indicate that Ampelomyces strains occupy a niche in the phyllosphere where they act primarily as mycoparasites of powdery mildews. Our work has established a framework for a molecular genetic toolbox for the genus Ampelomyces using ATMT.

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ON THE COVER: [Benthic unicells plus abandoned cell walls following zoospore release of the new marine, sand‐dwelling genus Chrysoparadoxa australica gen. et sp. nov. (Chrysoparadoxophyceae) photographed with differential interference contrast optics. Two chloroplasts are U‐shaped at the ends of the cells and then split into 2 lobes. Ultrastructure shows the chloroplasts to be surrounded by only 2 membranes, a novel feature within the heterokonts. Photo credit: R Wetherbee. Vol. 55, No. 2, pp. 257–278]

Cell Membrane Tracking in Living Brain Tissue Using Differential Interference Contrast Microscopy.

Differential interference contrast (DIC) microscopy is widely used for observing unstained biological samples that are otherwise optically transparent. Combining this optical technique with machine vision could enable the automation of many life science experiments; however, identifying relevant features under DIC is challenging. In particular, precise tracking of cell boundaries in a thick ( ) slice of tissue has not previously been accomplished. We present a novel deconvolution algorithm that achieves the state-of-the-art performance at identifying and tracking these membrane locations. Our proposed algorithm is formulated as a regularized least squares optimization that incorporates a filtering mechanism to handle organic tissue interference and a robust edge-sparsity regularizer that integrates dynamic edge tracking capabilities. As a secondary contribution, this paper also describes new community infrastructure in the form of a MATLAB toolbox for accurately simulating DIC microscopy images of in vitro brain slices. Building on existing DIC optics modeling, our simulation framework additionally contributes an accurate representation of interference from organic tissue, neuronal cell-shapes, and tissue motion due to the action of the pipette. This simulator allows us to better understand the image statistics (to improve algorithms), as well as quantitatively test cell segmentation and tracking algorithms in scenarios, where ground truth data is fully known.

Can intracytoplasmic Morphologically Selected Spermatozoa Injection be Used as First Choice of Treatment for Severe Male Factor Infertility Patients?

Aim:This study was carried out to assess the outcome of the intracytoplasmic morphologically selected sperm injection (IMSI) technique compared with the previous failed intracytoplasmic sperm injection (ICSI) attempts in oligoasthenoteratozoospermia (OAT)/severe OAT (SOAT)/teratozoospermia patients.Setting:Institution-based, in vitro fertilization center.Study Design:It was a nonrandomized prospective study including 57 couples who had previous one or two ICSI failures (failure due to no implantation as embryos were transferred in these cycles and had no pregnancy) due to male factor. There was no case of total fertilization failure. IMSI was carried out in these couples and the results were compared with their previously failed ICSI attempts.Materials and Methods:Real-time selection of sperms was done using IMSI as it allows the assessment of fine nuclear morphology and vacuoles in the sperm head at a high magnification (>6000×) with differential interference contrast optics. Therefore, IMSI was applied in couples having OAT, SOAT or teratozoospermia as male factor and the results were compared with their previous failed ICSI attempts. Statistical analysis was carried out using GraphPad Prism.Results and Conclusion:Data analysis demonstrated significant difference in the fertilization rate between IMSI and previous ICSI attempts of these patients (30.0% vs. 52.0%; P < 0.05). The embryo quality, implantation and pregnancy rates with IMSI were also significantly higher than those of their previous ICSI cycles (32% vs. 56.4%; 30.2% vs. 68.5%; 0.0% vs. 62.4%; P < 0.05). Our conclusion is that the IMSI procedure improved embryo development and the clinical outcomes in the same infertile couples with male infertility and poor embryo development over their previous ICSI attempts and can be taken up as the treatment of choice in cases of severe male factor infertility.

Focal Macropatch Recordings of Synaptic Currents from the Drosophila Larval Neuromuscular Junction.

Drosophila neuromuscular junction (NMJ) is an excellent model system to study glutamatergic synaptic transmission. We describe the technique of focal macropatch recordings of synaptic currents from visualized boutons at the Drosophila larval NMJ. This technique requires customized fabrication of recording micropipettes, as well as a compound microscope equipped with a high magnification, long-distance water immersion objective, differential interference contrast (DIC) optics, and a fluorescent attachment. The recording electrode is positioned on the top of a selected synaptic bouton visualized with DIC optics, epi-fluorescence, or both. The advantage of this technique is that it allows monitoring the synaptic activity of a limited number of sites of release. The recording electrode hasa diameter of several microns, and the release sites positioned outside of the electrode rim donot significantly affect the recorded currents. The recorded synaptic currents have fast kinetics and can be readily resolved. These advantages are especially important for the studies of mutant fly lines with enhanced spontaneous or asynchronous synaptic activity.

Chiral supramolecular order revealed during the formation of calf thymus and phage DNA crystals

The control of DNA packaging has been reported to be dependent on an ordered liquid-crystalline state. However, the textural characteristics that are typical of crystals and that resemble mesophases have not been reported for highly polymerized or even shorter types of DNA filaments under in vitro conditions that favor crystallization. Because DNA crystals are expected to exhibit particular textural optical anisotropies, pure and highly polymerized calf thymus DNA and simpler λ phage DNA were crystallized from solution drops and were analyzed using high-performance polarization microscopy with and without differential interference contrast (DIC) optics. Both types of DNA formed crystals that exhibited chiral supramolecular textures resembling the twist-grain boundary (TGB) columnar mesophases described for liquid crystals and exhibited intrinsic negative birefringence. To the best of our knowledge, this is the first observation using polarization/interference optics of pure DNA crystals that have TGB columnar mesophase-like textural characteristics. A comparison of the crystals formed from the highly polymerized calf thymus DNA and those formed from the shorter phage DNA strands revealed textural differences. Compared to the phage DNA crystals, the crystals of highly polymerized thymus DNA exhibited a more intertwisted columnar distribution and a fibrous texture between their columnar structures. In addition, a form birefringence phenomenon was detected only in the thymus DNA crystals. These characteristics are presumed to reflect the higher level of supramolecular order, self-assembly and chirality in highly polymerized calf thymus DNA crystals relative to that of crystals formed from the simpler, shorter, λ phage DNA. The higher-order supramolecular organization revealed here for in vitro DNA preparations raises the possibility that this structure could also occur, possibly to a smaller degree, during DNA self-aggregation under specific in vivo conditions. Whether the DNA crystal properties presently described play a role in the establishment of higher–order levels of hierarchical chromatin structure and consequently in chromatin physiology, should be further investigated.

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ON THE COVER: Filaments of the new genus Kraftionema allantoideum photographed with differential interference contrast optics. Mature fi laments on the left are comprised of allantoid cells or bicells arranged loosely within a capsule, appearing as a string of sausages. At the center of the image, a bicell has divided to produce 4 cells with abutting walls. Large, distinctive pyrenoids with surrounding starch sheaths are obvious. Photo credit: R. Wetherbee [Vol. 52, No. 5, pp. 704–715]

Staying connected: structural integration at the intervertebral disc-vertebra interface of human lumbar spines.

To investigate the microscopic fibrous integration between the intervertebral disc, cartilage endplates and vertebral endplates in human lumbar spines of varying degrees of degeneration using differential interference contrast (DIC) optics. Weakness at these junctions is considered to be an important factor in the aetiology of disc herniations. Magnetic resonance images (MRIs) of cadaveric lumbar spines were graded for degeneration and motion segments from a range of degenerative grades isolated and bisected sagittally. Following fixation and decalcification, these were cut into segments containing anterior or posterior annulus fibrosus or nucleus pulposus. The segments were cryo-sectioned and sections visualised using both standard light and DIC microscopy. Detachment at the interface between the disc and vertebrae increased with greater degenerative grade (from 1.9% in Grade I to 28% in Grade V), especially at the boundary between the cartilage and vertebral endplates. DIC microscopy revealed the fibrous organisation at the IVD-cartilage endplate interface with structural features, such as annular lamellae branching and nodal insertions in the nucleus pulposus region; these have been previously observed in ovine spines, but were less uniform in humans. Structural integrity of the IVD and cartilage endplate was also lost with increasing degeneration. This preliminary study shows that microscopic structural features may act to maintain attachment between the IVD and CEP in the human spine. Loss of structural integrity in this region may destabilise the spine, possibly altering the mechanical environment of the cells in the disc and so potentially contribute to the aetiopathogenesis of IVD degeneration.

English

Microalgae of benthic origins are abundant in aquatic ecosystems which have been affected by anthropogenic factors and provide a valuable aid in determining water quality. This study was initiated to understand the changes that occurred in the microphytobenthos diatom community in the pristine Mnweni river system in the KwaZulu-Natal Drakensberg region. Diatom frustules were examined under an Olympus BX41TF light microscope with Differential Interference Contrast (DIC) optics. The PRIMER v5 computer software package was utilised to perform multivariate data analyses which included clustering, ordination and k-dominance curves. The diatom Cymbella turgidula was present in all five sites whilst Encyonopsis stafsholtii was abundant in sites three and four. A SIMPER analysis of the data showed an average dissimilarity between the sites. Sites with higher total dissolved solids (TDS), phosphorous and nitrogen showed suppressed pH values although all values were above seven. The diatom composition together with environmental gradients in the catchment suggests that river chemistry, environment structure, current pattern and terrestrial use had important impacts on the integrity of the system. The diatom species that were found in the study sites suggested that the water in the Mnweni catchment was clean or mildly enriched. © 2016 Friends Science Publishers

Observation of the Ciliary Movement of Choroid Plexus Epithelial Cells Ex Vivo.

The choroid plexus is located in the ventricular wall of the brain, the main function of which is believed to be production of cerebrospinal fluid. Choroid plexus epithelial cells (CPECs) covering the surface of choroid plexus tissue harbor multiple unique cilia, but most of the functions of these cilia remain to be investigated. To uncover the function of CPEC cilia with particular reference to their motility, an ex vivo observation system was developed to monitor ciliary motility during embryonic, perinatal and postnatal periods. The choroid plexus was dissected out of the brain ventricle and observed under a video-enhanced contrast microscope equipped with differential interference contrast optics. Under this condition, a simple and quantitative method was developed to analyze the motile profiles of CPEC cilia for several hours ex vivo. Next, the morphological changes of cilia during development were observed by scanning electron microscopy to elucidate the relationship between the morphological maturity of cilia and motility. Interestingly, this method could delineate changes in the number and length of cilia, which peaked at postnatal day (P) 2, while the beating frequency reached a maximum at P10, followed by abrupt cessation at P14. These techniques will enable elucidation of the functions of cilia in various tissues. While related techniques have been published in a previous report(1), the current study focuses on detailed techniques to observe the motility and morphology of CPEC cilia ex vivo.