Morphophysiological Changes in Staphylococcus aureus Biofilms Treated with Plasma-Activated Hydrogen Peroxide Solution

Plasma-activated solution has attracted more attention in the food industry due to no chemical residue and good bacteriostatic properties. This study aimed to evaluate the effects of plasma-activated hydrogen peroxide solution (PAH) on the morphophysiology of Staphylococcus aureus biofilms. PAH was prepared using dielectric-barrier-discharge plasma and incubated with S. aureus biofilms for 0–40 min. Changes in biofilm morphophysiology were evaluated with laser scanning confocal microscopy, electron microscopic images, reactive oxygen species (ROS) content, metabolic capacity, and 1% agarose gel. Results indicated that the population of S. aureus in the biofilms was reduced by 4.04-log after incubation with PAH for 30 min. The thickness and metabolic capacity of biofilms were decreased, the ROS content and DNA fragments of bacteria increased after PAH treatments. Data suggested that PAH treatments significantly destroyed the morphophysiology of S. aureus (ATCC 6538) biofilms and could be considered as a valuable anti-biofilm technology to reduce foodborne pathogens on food and/or in food facilities.

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Cytokinin- and auxin-induced stomatal opening involves a decrease in levels of hydrogen peroxide in guard cells of Vicia faba

Functional Plant Biology ◽

10.1071/fp05232 ◽

2006 ◽

Vol 33 (6) ◽

pp. 573 ◽

Cited By ~ 34

Author(s):

Xi-Gui Song ◽

Xiao-Ping She ◽

Jun-Min He ◽

Chen Huang ◽

Tu-sheng Song

Keyword(s):

Hydrogen Peroxide ◽

Vicia Faba ◽

Laser Scanning ◽

Guard Cells ◽

Laser Scanning Confocal Microscopy ◽

Stomatal Opening ◽

Scanning Confocal Microscopy ◽

Diphenylene Iodonium ◽

The Relationship ◽

Exogenous H2o2

Previous studies have shown that cytokinins and auxins can induce the opening of stomata. However, the mechanism of stomatal opening caused by cytokinins and auxins remains unclear. The purpose of this paper is to investigate the relationship between hydrogen peroxide (H2O2) levels in guard cells and stomatal opening induced by cytokinins and auxins in Vicia faba. By means of stomatal bioassay and laser-scanning confocal microscopy, we provide evidence that cytokinins and auxins reduced the levels of H2O2 in guard cells and induced stomatal opening in darkness. Additionally, cytokinins not only reduced exogenous H2O2 levels in guard cells caused by exposure to light, but also abolished H2O2 that had been generated during a dark period, and promoted stomatal opening, as did ascorbic acid (ASA, an important reducing substrate for H2O2 removal). However, unlike cytokinins, auxins did not reduce exogenous H2O2, did not abolish H2O2 that had been generated in the dark, and therefore did not promote reopening of stoma induced to close in the dark. The above-mentioned effects of auxins were similar to that of diphenylene iodonium (DPI, an inhibitor of the H2O2-generating enzyme NADPH oxidase). Taken together our results indicate that cytokinins probably reduce the levels of H2O2 in guard cells by scavenging, whereas auxins limit H2O2 levels through restraining H2O2 generation, inducing stomatal opening in darkness.

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A Rapid Method for Combined Laser Scanning Confocal Microscopic and Electron Microscopic Visualization of Biocytin or Neurobiotin-labeled Neurons

Journal of Histochemistry & Cytochemistry ◽

10.1177/002215549804600216 ◽

1998 ◽

Vol 46 (2) ◽

pp. 263-273 ◽

Cited By ~ 10

Author(s):

Xue J. Sun ◽

Leslie P. Tolbert ◽

John G. Hildebrand ◽

Ian A. Meinertzhagen

Keyword(s):

Confocal Microscopy ◽

Laser Scanning ◽

Fluorescent Dyes ◽

Laser Scanning Confocal Microscopy ◽

Single Step ◽

Three Dimensions ◽

Fluorescent Label ◽

Electron Microscopic ◽

Scanning Confocal Microscopy ◽

Long Term Storage

Intracellular recording and dye filling are widely used to correlate the morphology of a neuron with its physiology. With laser scanning confocal microscopy, the complex shapes of labeled neurons in three dimensions can be reconstructed rapidly, but this requires fluorescent dyes. These dyes are neither permanent nor electron dense and therefore do not allow investigation by electron microscopy. Here we report a technique that quickly and easily converts a fluorescent label into a more stable and electron-dense stain. With this technique, a neuron is filled with Neurobiotin or biocytin, reacted with fluorophore-conjugated avidin, and imaged by confocal microscopy. To permit long-term storage or EM study, the fluorescent label is then converted to a stable electron-dense material by a single-step conversion using a commercially available ABC kit. We find that the method, which apparently relies on recognition of avidin's excess biotin binding sites by the biotin–peroxidase conjugate, is both faster and less labor intensive than photo-oxidation procedures in common use. The technique is readily adaptable to immunocytochemistry with biotinylated probes, as we demonstrate using anti-serotonin as an example.

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Use of Fluorescent Microspheres as a Tool To Investigate Bacterial Interactions with Growing Plants

Journal of Food Protection ◽

10.4315/0362-028x-68.4.870 ◽

2005 ◽

Vol 68 (4) ◽

pp. 870-873 ◽

Cited By ~ 29

Author(s):

ETHAN B. SOLOMON ◽

KARL R. MATTHEWS

Keyword(s):

Foodborne Pathogens ◽

Laser Scanning ◽

Leaf Tissue ◽

Laser Scanning Confocal Microscopy ◽

Escherichia Coli O157 ◽

Fluorescent Microspheres ◽

E Coli ◽

Bacterial Interactions ◽

Scanning Confocal Microscopy ◽

Tissue Sections

Foodborne pathogens may exist as endophytes of growing plants. The internalization of Escherichia coli O157:H7 or other foodborne pathogens in growing lettuce plants may be independent of microbial factors. Mature lettuce plants were surface irrigated with E. coli O157:H7 or with FluoSpheres (fluorescent microspheres) and harvested 1, 3, and 5 days post-exposure. FluoSpheres were utilized as a bacterial surrogate. Microscopic examination of root, stem, and leaf tissue sections revealed that FluoSpheres were internalized into growing plants. Laser scanning confocal microscopy revealed that FluoSpheres were present within the root tissue and leaf stem tissue. The presence of FluoSpheres in internal portions of stem and leaf tissue suggests transport of the spheres from the root upward into the edible tissue. The level of uptake of FluoSpheres and E. coli O157:H7 was quantified using filtration. Numbers of FluoSpheres and E. coli O157:H7 cells in plant tissue were similar. The entry of E. coli O157:H7 into lettuce plants may be a passive event because the concentration of FluoSpheres was similar to that of the pathogen.

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Penicillium digitatum Suppresses Production of Hydrogen Peroxide in Host Tissue During Infection of Citrus Fruit

Phytopathology ◽

10.1094/phyto-97-11-1491 ◽

2007 ◽

Vol 97 (11) ◽

pp. 1491-1500 ◽

Cited By ~ 115

Author(s):

D. Macarisin ◽

L. Cohen ◽

A. Eick ◽

G. Rafael ◽

E. Belausov ◽

...

Keyword(s):

Hydrogen Peroxide ◽

Laser Scanning ◽

Citrus Fruit ◽

Laser Scanning Confocal Microscopy ◽

Host Tissue ◽

Penicillium Digitatum ◽

Penicillium Expansum ◽

Control Value ◽

Scanning Confocal Microscopy ◽

Production Of Hydrogen

During the infection of citrus fruit by Penicillium digitatum there is little evidence of a host defense response. This suggests that P. digitatum has the ability to suppress host defenses. The current study demonstrates that P. digitatum suppresses a defense-related hydrogen peroxide (H2O2) burst in host tissue. In contrast, the nonhost pathogen, Penicillium expansum, triggers production of a significant amount of H2O2 in citrus fruit exocarp. Using laser scanning confocal microscopy, we demonstrated that P. digitatum suppressed an elevation in H2O2 up to 42 h after inoculation. Nevertheless, H2O2 levels around wounds inoculated with P. expansum increased by 63-fold above the control. P. digitatum continued to suppress H2O2 production in citrus fruit exocarp up to 66 h postinoculation and H2O2 levels were actually threefold below that of noninoculated controls. In contrast, the H2O2 level was still about 11-fold above the control value in wound sites inoculated with P. expansum. Studies on the effect of organic acids (as pH modulators) on the response of citrus fruit to compatible and noncompatible pathogens indicated that pathogenicity was enhanced only when host-tissue acidification was accompanied by the suppression of H2O2. Additionally, pathogenicity of both P. digitatum and P. expansum on citrus fruit was significantly enhanced by the H2O2-scavenging enzyme catalase. Based on our study and previous reports regarding the potential involvement of citric acid and catalase in green mold pathogenesis, we suggest that these compounds are strongly associated with the virulence of P. digitatum.

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Using laser scanning confocal microscopy as a guide for electron microscopic study: a simple method for correlation of light and electron microscopy.

Journal of Histochemistry & Cytochemistry ◽

10.1177/43.3.7868862 ◽

1995 ◽

Vol 43 (3) ◽

pp. 329-335 ◽

Cited By ~ 38

Author(s):

X J Sun ◽

L P Tolbert ◽

J G Hildebrand

Keyword(s):

Electron Microscopy ◽

Confocal Microscopy ◽

Microscopic Study ◽

Electron Microscopic Study ◽

Laser Scanning ◽

Laser Scanning Confocal Microscopy ◽

Synaptic Connections ◽

Electron Microscopic ◽

Simple Method ◽

Scanning Confocal Microscopy

Anatomic study of synaptic connections in the nervous system is laborious and difficult, especially when neurons are large or have fine branches embedded among many other processes. Although electron microscopy provides a powerful tool for such study, the correlation of light microscopic appearance and electron microscopic detail is very time-consuming. We report here a simple method combining laser scanning confocal microscopy and electron microscopy for study of the synaptic relationships of the neurons in the antennal lobe, the first central neuropil in the olfactory pathway, of the moth Manduca sexta. Neurons were labeled intracellularly with neurobiotin or biocytin, two widely used stains. The tissue was then sectioned on a vibratome and processed with both streptavidin-nanogold (for electron microscopic study) and streptavidin-Cy3 (for confocal microscopic study) and embedded in epon/araldite. Interesting areas of the labeled neuron were imaged in the epon/araldite blocks with laser scanning confocal microscopy and then thin-sectioned at the indicated depth for electron microscopic study. This method provides an easy, reliable way to correlate three-dimensional light microscopic information with electron microscopic detail, and can be very useful in studies of synaptic connections.

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

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100158376 ◽

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.

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

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100148642 ◽

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.

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

Pharmaceutics ◽

10.3390/pharmaceutics13060861 ◽

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.

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

Macromolecules ◽

10.1021/ma010190d ◽

2001 ◽

Vol 34 (15) ◽

pp. 5186-5191 ◽

Cited By ~ 11

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

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

Journal of Histochemistry & Cytochemistry ◽

10.1177/42.11.7930524 ◽

1994 ◽

Vol 42 (11) ◽

pp. 1413-1416 ◽

Cited By ~ 24

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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