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Differential Interference Contrast Microscopy (differential + interference_contrast_microscopy)
Selected AbstractsElectroporation as a tool to study in vivo spinal cord regenerationDEVELOPMENTAL DYNAMICS, Issue 2 2003K. Echeverri Abstract Tailed amphibians such as axolotls and newts have the unique ability to fully regenerate a functional spinal cord throughout life. Where the cells come from and how they form the new structure is still poorly understood. Here, we describe the development of a technique that allows the visualization of cells in the living animal during spinal cord regeneration. A microelectrode needle is inserted into the lumen of the spinal cord and short rapid pulses are applied to transfer the plasmids encoding the green or red fluorescent proteins into ependymal cells close to the plane of amputation. The use of small, transparent axolotls permits imaging with epifluorescence and differential interference contrast microscopy to track the transfected cells as they contribute to the spinal cord. This technique promises to be useful in understanding how neural progenitors are recruited to the regenerating spinal cord and opens up the possibility of testing gene function during this process. Developmental Dynamics 226:418,425, 2003. © 2003 Wiley-Liss, Inc. [source] Use of episcopic differential interference contrast microscopy to identify bacterial biofilms on salad leaves and track colonization by Salmonella ThompsonENVIRONMENTAL MICROBIOLOGY, Issue 4 2008J. C. Warner Summary Zoonotic pathogens such as Salmonella can cause gastrointestinal illness if they are ingested with food. Foods such as salads pose a greater risk because they are consumed raw and have been the source of major outbreaks of disease from fresh produce. The novel light microscopy methods used in this study allow detailed, high resolution imaging of the leaf surface environment (the phyllosphere) and allow pathogen tracking. Episcopic differential interference contrast microscopy coupled with epifluorescence was used to view the natural microflora in situ on salad leaves and their topographical distribution. Fluorescent nucleic acid staining was used to differentiate between bacterial colonists and inorganic debris. Salmonella enterica serovar Thompson expressing green fluorescent protein was inoculated onto individual spinach leaves for 24 h at 22°C in order to observe spatial and temporal patterning of colonization on the two surfaces of each leaf under different osmotic conditions. The results obtained show that salad leaves are host to high numbers of bacteria, typically 105 per square millimetre. Cells are present in complex three-dimensional aggregations which often have a slimy appearance, suggesting the presence of biofilms. Washing of the leaves had little effect on the number of adherent pathogens, suggesting very strong attachment. Episcopic differential interference contrast microscopy is a rapid alternative to both scanning electron microscopy and confocal laser scanning microscopy for visualizing leaf topography and biofilm formation in the natural state. [source] Application of laser scanning cytometry followed by epifluorescent and differential interference contrast microscopy for the detection and enumeration of Cryptosporidium and Giardia in raw and potable watersJOURNAL OF APPLIED MICROBIOLOGY, Issue 4 2002M.-R. De Roubin Aims: The main goal of this study was to validate a new laser scanning cytometry method (ChemScanRDI) that couples immunofluorescence detection with differential interference contrast (DIC) confirmation, against manual microscopic enumeration of Giardia and Cryptosporidium (oo)cysts. This study also assessed the basic performance of the new Association Française de Normalisation (AFNOR) NF T 90-455 method for Giardia and Cryptosporidium (oo)cyst enumeration with respect to (oo)cyst yield, linearity, repeatability, influence of turbidity and detection limit in raw and potable waters. Methods and Results: The new standard method relies on cartridge (Envirocheck) filtration, immunomagnetic separation purification, immunofluorescence staining and detection followed by DIC confirmation. The recovery was 30,50% for both parasites at seeding levels from 30 to 230 (oo)cysts. The method is linear from 0 to around 400 seeded (oo)cysts and the yield does not significantly vary for turbidity levels from 10 to 40 Formazin Nephelometric Units (FNU). The results were obtained using manual microscopic enumeration of the (oo)cysts. The ChemScanRDI yielded counts that were at least equivalent to those obtained using manual microscopy for both parasites in raw and potable water concentrates, for seeding levels of 10,300 or 10,100, respectively. The purification and labelling method proposed by the supplier of theChemScanRDI (Chemunex) reached very similar recoveries to the AFNOR protocol (70,86% in both cases). Conclusions: Laser scanning cytometry can be used as a more standardized alternative to manual enumeration as part of the new AFNOR standard method. Significance and Impact of the Study: By using laser scanning cytometry instead of manual microscopy, laboratories could circumvent the limitations of manual microscopy, namely: low sample throughput, operator subjectivity and operator fatigue. The study further supports the drive to incorporate laser scanning cytometry in the standard methods for Giardia and Cryptosporidium enumeration. [source] Food uptake in the mixotrophic Dinophysis acuminataTHE JOURNAL OF EUKARYOTIC MICROBIOLOGY, Issue 2 2005LUCIE MARANDA Evidence of food uptake in the photosynthetic genus Dinophysis comes solely from the presence of food vacuoles, as no photosynthetic cells have ever been observed in the act of feeding. We examined the feeding ecology of D. acuminata in natural populations and under laboratory conditions. Using depth-integrated sampling of the water column, we determined the frequency of food vacuolated cells at 2-h intervals over a 24-h period in a shallow marine embayment. Food vacuoles in preserved cells were enumerated using Nomarski differential interference contrast microscopy; ultrastructural characters were recorded by transmission electron microscopy. A peak in the feeding activity was observed toward dusk for an abundant June population, with 26% of cells with at least one food vacuole. Mechanisms of concurrent carbon acquisition were evident from the presence of chloroplasts with starch grains and food vacuoles within the same cell. Vacuole content could not be identified. In a preliminary 2-wk long simulated grazing experiment, a mixture of two hypothesized preys, Rhodomonas salina and Dunaliella tertiolecta, was offered to D. acuminata; the Dinophysis populations decreased steadily and at the same rate, whether food was present or not. The evaluation of the food vacuole frequency will be repeated in the coming season to verify the observed pattern, while grazing experiments will include a variety of food items and incubation conditions. Our current inability to successfully culture any photosynthetic Dinophysis limits ecophysiological approaches, either at the population or cellular level, to manipulation of field samples. Supported by National Institutes of Health Grant GM62126-01A1. [source] | ||||||||||