scholarly journals Prototheca spp. induce an inflammatory response via mtROS-mediated activation of NF-κB and NLRP3 inflammasome pathways in bovine mammary epithelial cell cultures

2021 ◽  
Vol 52 (1) ◽  
Author(s):  
Wenpeng Zhao ◽  
Fumeng He ◽  
Herman W. Barkema ◽  
Siyu Xu ◽  
Jian Gao ◽  
...  
Keyword(s):  
Nlrp3 Inflammasome ◽  
Laser Scanning ◽  
Inflammatory Responses ◽  
Bovine Mastitis ◽  
Laser Scanning Confocal Microscopy ◽  
Mammary Epithelial ◽  
Mitochondrial Activity ◽  
Severe Reaction ◽  
Scanning Confocal Microscopy ◽  
Transmission Electron

AbstractEmergence of bovine mastitis caused by Prototheca algae is the impetus to better understand these infections. Both P. bovis and P. ciferrii belong to Prototheca algae, but they differ in their pathogenicity to induce inflammatory responses. The objective was to characterize and compare pathogenesis of inflammatory responses in bMECs induced by P. bovis versus P. ciferrii. Mitochondrial ultrastructure, activity and mtROS in bMECs were assessed with transmission electron microscopy and laser scanning confocal microscopy. Cytokines, including TNF-α, IL-1β and IL-18, were measured by ELISA and real-time PCR, whereas expressions of various proteins in the NF-κB and NLRP3 inflammasome pathways were detected with immunofluorescence or Western blot. Infection with P. bovis or P. ciferrii damaged mitochondria, including dissolution and vacuolation of cristae, and decreased mitochondrial activity, with P. bovis being more pathogenic and causing greater destruction. There were increases in NADPH production and mtROS accumulation in infected bMECs, with P. bovis causing greater increases and also inducing higher cytokine concentrations. Expressions of NF-κB-p65, p-NF-κB-p65, IκBα and p-IκBα proteins in the NF-κB pathway, as well as NLRP3, Pro Caspase1, Caspase1 p20, ASC, Pro IL-1β, and IL-1β proteins in the NLRP3 inflammasome pathway, were significantly higher in P. bovis-infected bMECs. However, mito-TEMPO significantly inhibited production of cytokines and decreased expression of proteins in NF-κB and NLRP3 inflammasome pathways in bMECs infected with either P. bovis or P. ciferrii. In conclusion, P. bovis or P. ciferrii infections induced inflammatory responses in bMECs, with increased mtROS in damaged mitochondria and activated NF-κB and NLRP3 inflammasome pathways, with P. bovis causing a more severe reaction.

Morphology of antennular sensors in Clausocalanus furcatus (Copepoda: Calanoida)

2008 ◽  
Vol 88 (3) ◽  
pp. 535-541 ◽  
Author(s):  
Marco Uttieri ◽  
Euan R. Brown ◽  
Geoff A. Boxshall ◽  
Maria Grazia Mazzocchi
Keyword(s):  
Laser Scanning ◽  
Three Dimensional ◽  
Laser Scanning Confocal Microscopy ◽  
Specific Aspect ◽  
Dual Function ◽  
Aquatic Environments ◽  
Scanning Confocal Microscopy ◽  
Transmission Electron ◽  
Sensory Structures ◽  
Clausocalanus Furcatus

The success of planktonic copepods in aquatic environments is dependent on efficient sensing of their three-dimensional surroundings. The detection of external cues is of importance for the localization of other organisms (prey, predators and mates), and is mediated by an array of mechano- and chemoreceptors located on the paired antennules (A1).We investigated the morphology and distribution pattern of A1 sensory structures in the adult female of Clausocalanus furcatus (Copepoda: Calanoida) using different techniques (camera lucida, scanning and transmission electron microscopy and laser scanning confocal microscopy) each focusing on a specific aspect of the structures analysed. Integration of the information collected shows that C. furcatus possesses an array of mechanical, chemical and dual-function sensors over its A1, by which the copepod can detect different stimuli from the environment. Results are discussed in the light of the unique swimming behaviour displayed by this widespread epipelagic copepod.

Ultrastructural Analysis of Healthy Synovial Fluids in Three Mammalian Species

2014 ◽  
Vol 20 (3) ◽  
pp. 903-911 ◽  
Author(s):  
Constantin I. Matei ◽  
Caroline Boulocher ◽  
Christelle Boulé ◽  
Michael Schramme ◽  
Eric Viguier ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Transmission Electron Microscopy ◽  
Laser Scanning ◽  
Mammalian Species ◽  
Structural Features ◽  
Laser Scanning Confocal Microscopy ◽  
Ultrastructural Organization ◽  
Synovial Joint ◽  
Scanning Confocal Microscopy ◽  
Transmission Electron

AbstractA better knowledge of synovial fluid (SF) ultrastructure is required to further understand normal joint lubrication and metabolism. The aim of the present study was to elucidate SF structural features in healthy joints from three mammalian species of different size compared with features in biomimetic SF. High-resolution structural analysis was performed using transmission electron microscopy (TEM) and scanning electron microscopy (SEM) and environmental SEM/wet scanning transmission electron microscopy mode complemented by TEM and SEM cryogenic methods. Laser-scanning confocal microscopy (LCM) was used to locate the main components of SF with respect to its ultrastructural organization. The present study showed that the ultrastructure of healthy SF is built from a network of vesicles with a size range from 100 to a few hundred nanometers. A multilayered organization of the vesicle membranes was observed with a thickness of about 5 nm. LCM study of biological SF compared with synthetic SF showed that the microvesicles consist of a lipid-based membrane enveloping a glycoprotein gel. Thus, healthy SF has a discontinuous ultrastructure based on a complex network of microvesicles. This finding offers novel perspectives for the diagnosis and treatment of synovial joint diseases.

scholarly journals Streamlined Embedding of Cell Monolayers on Gridded Glass-Bottom Imaging Dishes for Correlative Light and Electron Microscopy

2010 ◽  
Vol 16 (6) ◽  
pp. 747-754 ◽  
Author(s):  
Hugo H. Hanson ◽  
James E. Reilly ◽  
Rebecca Lee ◽  
William G. Janssen ◽  
Greg R. Phillips
Keyword(s):  
Electron Microscopy ◽  
Laser Scanning ◽  
Fluorescent Protein ◽  
Light And Electron Microscopy ◽  
Laser Scanning Confocal Microscopy ◽  
Step Method ◽  
Cell Monolayers ◽  
Scanning Confocal Microscopy ◽  
Transmission Electron ◽  
Green Fluorescent

AbstractCorrelative light and electron microscopy (CLEM) has facilitated study of intracellular trafficking. Routine application of CLEM would be advantageous for many laboratories but previously described techniques are particularly demanding, even for those with access to laser scanning confocal microscopy (LSCM) and transmission electron microscopy (TEM). We describe streamlined methods for TEM of green fluorescent protein (GFP)-labeled organelles after imaging by LSCM using gridded glass bottom imaging dishes. GFP-MAP 1A/1B LC3 (GFP-LC3) transfected cells were treated with rapamycin, fixed and imaged by LSCM. Confocal image stacks were acquired enabling full visualization of each GFP-LC3 labeled organelle. After LSCM, cells were embedded for TEM using a simplified two step method that stabilizes the glass bottom such that the block can be separated from the glass by mild heating. All imaging and TEM processing are performed in the same dish. The LSCM imaged cells were relocated on the block and serial sectioned. Correlation of LSCM, DIC, and TEM images was facilitated by cellular landmarks. All GFP labeled structures were successfully reidentified and imaged by serial section TEM. This method could make CLEM more accessible to nonspecialized laboratories with basic electron microscopy expertise and could be used routinely to confirm organelle localization of fluorescent puncta.

scholarly journals Interactions of Neisseria gonorrhoeae with Adherent Polymorphonuclear Leukocytes

2005 ◽  
Vol 73 (4) ◽  
pp. 1971-1977 ◽  
Author(s):  
Mark P. Simons ◽  
William M. Nauseef ◽  
Michael A. Apicella
Keyword(s):  
Neisseria Gonorrhoeae ◽  
Polymorphonuclear Leukocytes ◽  
Laser Scanning ◽  
Laser Scanning Confocal Microscopy ◽  
Intracellular Replication ◽  
Experimental Conditions ◽  
Scanning Confocal Microscopy ◽  
Transmission Electron ◽  
Large Numbers ◽  
Nadph Oxidase Activation

ABSTRACT Neisseria gonorrhoeae causes severe exudative urethritis. The exudates from infected individuals contain large numbers of polymorphonuclear leukocytes (PMN) with ingested gonococci. The fate of N. gonorrhoeae within PMN has been a topic of debate for years. In this study, we examined the interactions of N. gonorrhoeae with PMN adherent to surfaces as a system that better models events during clinical disease. Using chemiluminescence to measure reactive oxygen species (ROS), we found that N. gonorrhoeae stimulated PMN to produce a respiratory burst. Different kinetics were seen when PMN were stimulated with opsonized zymosan particles. In addition, ROS were produced predominantly inside the PMN in response to gonococci. Laser scanning confocal microscopy and transmission electron microscopy showed that N. gonorrhoeae rapidly associated with PMN under these experimental conditions and was internalized. Some gonococci were cleared in the first 30 to 60 min after phagocytosis, but a majority of the population persisted for 6 h after phagocytosis. Quantification of viable organisms showed that a significant portion of the population resisted killing. The viability of this subpopulation remained unchanged for 2 h after phagocytosis. A significant increase of viable gonococci from 1 to 6 h was also observed, suggesting intracellular replication. Four different N. gonorrhoeae strains demonstrated the same capacity to resist PMN-mediated killing, whereas Escherichia coli was rapidly killed by PMN under the same conditions. Taken together, these findings suggest that a subpopulation of N. gonorrhoeae resists killing and replicates within PMN phagosomes in spite of NADPH oxidase activation.

Laser Scanning Confocal Microscopy and Three-Dimesnsional Reconstruction of the Mitotic Spindles of Unequally Dividing Embryonic Cells

1990 ◽  
Vol 48 (3) ◽  
pp. 166-167
Author(s):  
J. Holy ◽  
G. Schatten
Keyword(s):  
Confocal Microscopy ◽  
Mitotic Spindle ◽  
Laser Scanning ◽  
Three Dimensional ◽  
Laser Scanning Confocal Microscopy ◽  
Two Dimensions ◽  
Embryonic Cells ◽  
Mitotic Spindles ◽  
Cleavage Division ◽  
Scanning Confocal Microscopy

One of the classic limitations of light microscopy has been the fact that three dimensional biological events could only be visualized in two dimensions. Recently, this shortcoming has been overcome by combining the technologies of laser scanning confocal microscopy (LSCM) and computer processing of microscopical data by volume rendering methods. We have employed these techniques to examine morphogenetic events characterizing early development of sea urchin embryos. Specifically, the fourth cleavage division was examined because it is at this point that the first morphological signs of cell differentiation appear, manifested in the production of macromeres and micromeres by unequally dividing vegetal blastomeres.The mitotic spindle within vegetal blastomeres undergoing unequal cleavage are highly polarized and develop specialized, flattened asters toward the micromere pole. In order to reconstruct the three-dimensional features of these spindles, both isolated spindles and intact, extracted embryos were fluorescently labeled with antibodies directed against either centrosomes or tubulin.

Automated 3-D cell population analysis in thick tissue sections using laser-scanning confocal-microscopy data

1993 ◽  
Vol 51 ◽  
pp. 562-563
Author(s):  
Hakan Ancin
Keyword(s):  
Laser Scanning ◽  
Population Analysis ◽  
Three Dimensional ◽  
Large Data ◽  
Laser Scanning Confocal Microscopy ◽  
Tissue Slices ◽  
Scanning Confocal Microscopy ◽  
Tissue Sections ◽  
Spatially Adaptive ◽  
Microscopy Data

This paper presents methods for performing detailed quantitative automated three dimensional (3-D) analysis of cell populations in thick tissue sections while preserving the relative 3-D locations of cells. Specifically, the method disambiguates overlapping clusters of cells, and accurately measures the volume, 3-D location, and shape parameters for each cell. Finally, the entire population of cells is analyzed to detect patterns and groupings with respect to various combinations of cell properties. All of the above is accomplished with zero subjective bias.In this method, a laser-scanning confocal light microscope (LSCM) is used to collect optical sections through the entire thickness (100 - 500μm) of fluorescently-labelled tissue slices. The acquired stack of optical slices is first subjected to axial deblurring using the expectation maximization (EM) algorithm. The resulting isotropic 3-D image is segmented using a spatially-adaptive Poisson based image segmentation algorithm with region-dependent smoothing parameters. Extracting the voxels that were labelled as "foreground" into an active voxel data structure results in a large data reduction.

scholarly journals Advanced Static and Dynamic Fluorescence Microscopy Techniques to Investigate Drug Delivery Systems

Pharmaceutics ◽  
2021 ◽  
Vol 13 (6) ◽  
pp. 861
Author(s):  
Jacopo Cardellini ◽  
Arianna Balestri ◽  
Costanza Montis ◽  
Debora Berti
Keyword(s):  
Drug Delivery ◽  
Fluorescence Microscopy ◽  
Drug Delivery Systems ◽  
Laser Scanning ◽  
Super Resolution ◽  
Laser Scanning Confocal Microscopy ◽  
Delivery Systems ◽  
Scanning Confocal Microscopy ◽  
Biological Phenomena

In the past decade(s), fluorescence microscopy and laser scanning confocal microscopy (LSCM) have been widely employed to investigate biological and biomimetic systems for pharmaceutical applications, to determine the localization of drugs in tissues or entire organisms or the extent of their cellular uptake (in vitro). However, the diffraction limit of light, which limits the resolution to hundreds of nanometers, has for long time restricted the extent and quality of information and insight achievable through these techniques. The advent of super-resolution microscopic techniques, recognized with the 2014 Nobel prize in Chemistry, revolutionized the field thanks to the possibility to achieve nanometric resolution, i.e., the typical scale length of chemical and biological phenomena. Since then, fluorescence microscopy-related techniques have acquired renewed interest for the scientific community, both from the perspective of instrument/techniques development and from the perspective of the advanced scientific applications. In this contribution we will review the application of these techniques to the field of drug delivery, discussing how the latest advancements of static and dynamic methodologies have tremendously expanded the experimental opportunities for the characterization of drug delivery systems and for the understanding of their behaviour in biologically relevant environments.

Observation of Fine Structure in Bicontinuous Phase-Separated Domains of a Polymer Blend by Laser Scanning Confocal Microscopy

2001 ◽  
Vol 34 (15) ◽  
pp. 5186-5191 ◽  
Author(s):  
Hiroshi Jinnai ◽  
Hiroshi Yoshida ◽  
Kohtaro Kimishima ◽  
Yoshinori Funaki ◽  
Yoshitsugu Hirokawa ◽  
...  
Keyword(s):  
Fine Structure ◽  
Confocal Microscopy ◽  
Polymer Blend ◽  
Laser Scanning ◽  
Laser Scanning Confocal Microscopy ◽  
Scanning Confocal Microscopy

scholarly journals The DNA content of trophozoites and cysts of Giardia lamblia by microdensitometric quantitation of Feulgen staining and examination by laser scanning confocal microscopy.

1994 ◽  
Vol 42 (11) ◽  
pp. 1413-1416 ◽  
Author(s):  
S L Erlandsen ◽  
E M Rasch
Keyword(s):  
Confocal Microscopy ◽  
Genome Size ◽  
Dna Content ◽  
Giardia Lamblia ◽  
Laser Scanning ◽  
Laser Scanning Confocal Microscopy ◽  
Evolutionary Development ◽  
Scanning Confocal Microscopy ◽  
Dividing Cells ◽  
C Value

We investigated direct measurement of the DNA content of the parasitic intestinal flagellate Giardia lamblia through quantitation by Feulgen microspectrophotometry and also by visualization of Feulgen-stained DNA chromosomes within dividing cells by laser scanning confocal microscopy. Individual trophozoites of Giardia (binucleate) contained 0.144 +/- 0.018 pg of DNA/cell or 0.072 pg DNA/nucleus. Giardia lamblia cysts (quadranucleate) contained 0.313 +/- 0.003 pg DNA or 0.078 pg DNA/nucleus. The genome size (C) value per nucleus ranged between 6.5-7.1 x 10(7) BP for trophozoites and cysts, respectively. Confocal microscopic examination of Giardia trophozoites undergoing binary fission revealed five chromosome-like bodies within each nucleus. Further information about genome size and DNA content within different Giardia species may help to clarify the pivotal role of these primitive eukaryotic cells in evolutionary development.

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