scholarly journals Direct fluorescence imaging of lignocellulosic and suberized cell walls in roots and stems

AoB Plants ◽  
2020 ◽  
Vol 12 (4) ◽  
Author(s):  
Peter Kitin ◽  
Satoshi Nakaba ◽  
Christopher G Hunt ◽  
Sierin Lim ◽  
Ryo Funada
Keyword(s):  
Cell Walls ◽  
Laser Scanning ◽  
Cell Types ◽  
Plant Evolution ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Chemical Information ◽  
Wide Field ◽  
Tissue Preparation ◽  
Plant Structure

Abstract Investigating plant structure is fundamental in botanical science and provides crucial knowledge for the theories of plant evolution, ecophysiology and for the biotechnological practices. Modern plant anatomy often targets the formation, localization and characterization of cellulosic, lignified or suberized cell walls. While classical methods developed in the 1960s are still popular, recent innovations in tissue preparation, fluorescence staining and microscopy equipment offer advantages to the traditional practices for investigation of the complex lignocellulosic walls. Our goal is to enhance the productivity and quality of microscopy work by focusing on quick and cost-effective preparation of thick sections or plant specimen surfaces and efficient use of direct fluorescent stains. We discuss popular histochemical microscopy techniques for visualization of cell walls, such as autofluorescence or staining with calcofluor, Congo red (CR), fluorol yellow (FY) and safranin, and provide detailed descriptions of our own approaches and protocols. Autofluorescence of lignin in combination with CR and FY staining can clearly differentiate between lignified, suberized and unlignified cell walls in root and stem tissues. Glycerol can serve as an effective clearing medium as well as the carrier of FY for staining of suberin and lipids allowing for observation of thick histological preparations. Three-dimensional (3D) imaging of all cell types together with chemical information by wide-field fluorescence or confocal laser scanning microscopy (CLSM) was achieved.

Different patterns of rDNA distribution in Pisum sativum nucleoli correlate with different levels of nucleolar activity

1993 ◽  
Vol 104 (3) ◽  
pp. 843-852 ◽  
Author(s):  
M. I. Highett ◽  
D. J. Rawlins ◽  
P. J. Shaw
Keyword(s):  
Pisum Sativum ◽  
Laser Scanning ◽  
Three Dimensional ◽  
Ccd Camera ◽  
Cell Types ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Wide Field ◽  
Root Tips ◽  
Root Cells

We have used in situ hybridization with probes to rDNA, labelled either with digoxygenin or directly with fluorescein, to determine the arrangement of these genes within the nucleoli of Pisum sativum L. root cells. Confocal laser scanning microscopy was used to image the three-dimensional structures revealed, but we have also compared this technique with deconvolution of conventional (wide-field) fluorescence images measured with a cooled CCD camera, and have shown that the results are remarkably similar. When the deconvolution technique was applied to the confocal data it gave clearer images than could be achieved by confocal microscopy alone. We have analysed the distribution of rDNA in the different cell types observable in root tips: the quiescent centre; active meristematic cells; and relatively differentiated root cap, epidermal and cortical cells. In addition to four perinucleolar knobs of condensed, inactive rDNA genes, corresponding to the four nucleolar organizers in P. sativum, which were the most brightly labelled structures, several characteristic patterns of intranucleolar labelling were apparent, including bright foci, large central chromatin masses, and fine, decondensed interconnecting fibres. The larger and more active the nucleolus, the smaller the proportion of condensed perinucleolar rDNA. In some large and active meristematic nucleoli, all the internal rDNA is decondensed, showing that transcription cannot be restricted to the bright foci, and is most likely to occur on the decondensed fibres.

scholarly journals The MNN2 Gene Knockout Modulates the Antifungal Resistance of Biofilms of Candida glabrata

Biomolecules ◽  
2018 ◽  
Vol 8 (4) ◽  
pp. 130 ◽  
Author(s):  
Celia F. Rodrigues ◽  
Diana Vilas Boas ◽  
Ken Haynes ◽  
Mariana Henriques
Keyword(s):  
Cell Walls ◽  
Candida Glabrata ◽  
Laser Scanning ◽  
Reference Strain ◽  
Gene Knockout ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Drug Treatments ◽  
Biomass Reduction ◽  
Biofilm Matrix

Candida glabrata biofilms are recognized to have high resistance to antifungals. In order to understand the effect of mannans in the resistance profile of C. glabrata mature biofilms, C. glabrata Δmnn2 was evaluated. Biofilm cell walls were analysed by confocal laser scanning microscopy (CLSM) and their susceptibility was assessed for fluconazole, amphotericin B, caspofungin, and micafungin. Crystal violet and Alcian Blue methods were performed to quantify the biomass and the mannans concentration in the biofilm cells and matrices, respectively. The concentration of β-1,3 glucans was also measured. No visible differences were detected among cell walls of the strains, but the mutant had a high biomass reduction, after a drug stress. When compared with the reference strain, it was detected a decrease in the susceptibility of the biofilm cells and an increase of β-1,3 glucans in the C. glabrata Δmnn2. The deletion of the MNN2 gene in C. glabrata induces biofilm matrix and cell wall variabilities that increase the resistance to the antifungal drug treatments. The rise of β-1,3 glucans appears to have a role in this effect.

scholarly journals Assessment of Human Pancreatic Islet Architecture and Composition by Laser Scanning Confocal Microscopy

2005 ◽  
Vol 53 (9) ◽  
pp. 1087-1097 ◽  
Author(s):  
Marcela Brissova ◽  
Michael J. Fowler ◽  
Wendell E. Nicholson ◽  
Anita Chu ◽  
Boaz Hirshberg ◽  
...  
Keyword(s):  
Pancreatic Islet ◽  
Laser Scanning ◽  
Cell Types ◽  
Human Islets ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Human Pancreas ◽  
Pancreatic Islet Transplantation ◽  
Histological Sections ◽  
Human Primate

The recent success of pancreatic islet transplantation has generated considerable enthusiasm. To better understand the quality and characteristics of human islets used for transplantation, we performed detailed analysis of islet architecture and composition using confocal laser scanning microscopy. Human islets from six separate isolations provided by three different islet isolation centers were compared with isolated mouse and non-human primate islets. As expected from histological sections of murine pancreas, in isolated murine islets α and δ cells resided at the periphery of the β-cell core. However, human islets were markedly different in that α, β, and δ cells were dispersed throughout the islet. This pattern of cell distribution was present in all human islet preparations and islets of various sizes and was also seen in histological sections of human pancreas. The architecture of isolated non-human primate islets was very similar to that of human islets. Using an image analysis program, we calculated the volume of α, β, and δ cells. In contrast to murine islets, we found that populations of islet cell types varied considerably in human islets. The results indicate that human islets not only are quite heterogeneous in terms of cell composition but also have a substantially different architecture from widely studied murine islets.

Sterols and lignin in Eucalyptus globulus Labill. wood: Spatial distribution and fungal removal as revealed by microscopy and chemical analyses

Holzforschung ◽  
2009 ◽  
Vol 63 (3) ◽  
Author(s):  
Mariela Speranza ◽  
Ana Gutiérrez ◽  
José Carlos del Río ◽  
Lina Bettucci ◽  
Ángel T. Martínez ◽  
...  
Keyword(s):  
Eucalyptus Globulus ◽  
Laser Scanning ◽  
Cell Types ◽  
Laser Scanning Microscopy ◽  
Gas Chromatography Mass Spectrometry ◽  
Confocal Laser ◽  
Lipophilic Compounds ◽  
Parenchyma Cells ◽  
Ray Parenchyma ◽  
Ray Parenchyma Cells

AbstractWood decay experiments were carried out aiming at the selective removal of lipophilic compounds with selected basidiomycetes isolated fromEucalyptus globulusplantations in Uruguay:Dendrophora albobadia,Lentinus tigrinus,Peniophora cinerea,Peniophora lycii, andPhanerochaete crassa. Localization and composition of lipophilic compounds and lignin ofE. globuluswere determined by gas chromatography-mass spectrometry, fluorescence microscopy using filipin staining, confocal laser scanning microscopy (CLSM), and low temperature scanning electron microscopy. Free and esterified sterols, mainly sitosterol, were the predominant lipophilic compounds in the control wood. Sterols were present in ray parenchyma cells, together with polyphenols, and in vessels. This confirms earlier observations indicating that these cell types are the principal source of lipophilic extractives involved in pitch problems during pulping and bleaching. Sterols are also present in the vestures of fiber and vessel pits. Different fungal degradation patterns ofE. globuluswood were determined.P. lyciishowed the highest specificity for lignin degradation during short incubation time together with considerable sterol removal capacity. Ray parenchyma cells and their lumen deposits were strongly degraded byP. lycii. Eucalypt lignin located in vessel walls and fiber cell corners was more resistant to fungal attack, as revealed by CLSM. The initial decay stage ofL. tigrinuswas restricted to vessels and tyloses where the sterol compounds were removed.

scholarly journals Uptake of the proteins HTRA1 and HTRA2 by cells mediated by calcium phosphate nanoparticles

2017 ◽  
Vol 8 ◽  
pp. 381-393 ◽  
Author(s):  
Olga Rotan ◽  
Katharina N Severin ◽  
Simon Pöpsel ◽  
Alexander Peetsch ◽  
Melisa Merdanovic ◽  
...  
Keyword(s):  
Calcium Phosphate ◽  
Cell Membrane ◽  
Laser Scanning ◽  
Cell Types ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Synthetic Drugs ◽  
Uptake Pathway ◽  
Calcium Phosphate Nanoparticles ◽  
Dissolved Form

The efficient intracellular delivery of (bio)molecules into living cells remains a challenge in biomedicine. Many biomolecules and synthetic drugs are not able to cross the cell membrane, which is a problem if an intracellular mode of action is desired, for example, with a nuclear receptor. Calcium phosphate nanoparticles can serve as carriers for small and large biomolecules as well as for synthetic compounds. The nanoparticles were prepared and colloidally stabilized with either polyethyleneimine (PEI; cationic nanoparticles) or carboxymethyl cellulose (CMC; anionic nanoparticles) and loaded with defined amounts of the fluorescently labelled proteins HTRA1, HTRA2, and BSA. The nanoparticles were purified by ultracentrifugation and characterized by dynamic light scattering and scanning electron microscopy. Various cell types (HeLa, MG-63, THP-1, and hMSC) were incubated with fluorescently labelled proteins alone or with protein-loaded cationic and anionic nanoparticles. The cellular uptake was followed by light and fluorescence microscopy, confocal laser scanning microscopy (CLSM), and flow cytometry. All proteins were readily transported into the cells by cationic calcium phosphate nanoparticles. Notably, only HTRA1 was able to penetrate the cell membrane of MG-63 cells in dissolved form. However, the application of endocytosis inhibitors revealed that the uptake pathway was different for dissolved HTRA1 and HTRA1-loaded nanoparticles.

scholarly journals Morphometric studies of the localization of the glucocorticoid receptor in mammalian cells and of glucocorticoid hormone-induced effects.

1994 ◽  
Vol 42 (5) ◽  
pp. 645-657 ◽  
Author(s):  
G Akner ◽  
A C Wikström ◽  
K Mossberg ◽  
K G Sundqvist ◽  
J A Gustafsson
Keyword(s):  
Glucocorticoid Receptor ◽  
Mammalian Cells ◽  
Laser Scanning ◽  
Nuclear Translocation ◽  
Cell Types ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Mitotic Apparatus ◽  
Glucocorticoid Treatment ◽  
Cytoplasmic Microtubules

We studied the subcellular distribution of the glucocorticoid receptor (GR) by light microscopy (LM) and confocal laser scanning microscopy (CLSM) in different mammalian cell types. The effect of added glucocorticoid hormones on GR distribution was investigated by photometric quantitation on optical sections obtained by CLSM followed by statistical analysis. In the control interphase cytoplasm, the distribution of GR was fibrillar in some and diffuse in other cell types. Fibrillar GR was distributed along cytoplasmic microtubules (MTs) with predilection for a subset of MTs. GR was also observed in the centrosomes. Nuclear GR was both diffuse and granular in distribution. During cell division, GR appeared in the mitotic apparatus at all stages of mitosis. These findings were not fixation-dependent. Glucocorticoid treatment increased both the nuclear and cytoplasmic GR signal. However, this was detectable only after precipitating but not cross-linking fixation. There was both intra- and intercellular GR heterogeneity in the absence and presence of hormone but no indication of a hormone-induced nuclear translocation of GR. We present a hypothetical model of two independent GR populations in the nucleus and cytoplasm, respectively, without any discernible ligand-induced nuclear translocation of GR. The extranuclear GR population may exert effect(s) on site in the cytoplasm without involving nuclear genomic transcription.

scholarly journals Microscopic and Spectroscopic Analyses of Chlorhexidine Tolerance in Delftia acidovorans Biofilms

2014 ◽  
Vol 58 (10) ◽  
pp. 5673-5686 ◽  
Author(s):  
Tara Rema ◽  
John R. Lawrence ◽  
James J. Dynes ◽  
Adam P. Hitchcock ◽  
Darren R. Korber
Keyword(s):  
Laser Scanning ◽  
Cell Types ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Content Type ◽  
Flow Cells ◽  
Biofilm Thickness ◽  
Delftia Acidovorans ◽  
Scanning Transmission ◽  
Negative Effect

ABSTRACTThe physicochemical responses ofDelftia acidovoransbiofilms exposed to the commonly used antimicrobial chlorhexidine (CHX) were examined in this study. A CHX-sensitive mutant (MIC, 1.0 μg ml−1) was derived from a CHX-tolerant (MIC, 15.0 μg ml−1)D. acidovoransparent strain using transposon mutagenesis.D. acidovoransmutant (MT51) and wild-type (WT15) strain biofilms were cultivated in flow cells and then treated with CHX at sub-MIC and inhibitory concentrations and examined by confocal laser scanning microscopy (CLSM), scanning transmission X-ray microscopy (STXM), and infrared (IR) spectroscopy. Specific morphological, structural, and chemical compositional differences between the CHX-treated and -untreated biofilms of both strains were observed. Apart from architectural differences, CLSM revealed a negative effect of CHX on biofilm thickness in the CHX-sensitive MT51 biofilms relative to those of the WT15 strain. STXM analyses showed that the WT15 biofilms contained two morphochemical cell variants, whereas only one type was detected in the MT51 biofilms. The cells in the MT51 biofilms bioaccumulated CHX to a similar extent as one of the cell types found in the WT15 biofilms, whereas the other cell type in the WT15 biofilms did not bioaccumulate CHX. STXM and IR spectral analyses revealed that CHX-sensitive MT51 cells accumulated the highest levels of CHX. Pretreating biofilms with EDTA promoted the accumulation of CHX in all cells. Thus, it is suggested that a subpopulation of cells that do not accumulate CHX appear to be responsible for greater CHX resistance inD. acidovoransWT15 biofilm in conjunction with the possible involvement of bacterial membrane stability.

scholarly journals A Selective Ratiometric Fluorescent Probe for No-Wash Detection of PVC Microplastic

Polymers ◽  
2021 ◽  
Vol 13 (10) ◽  
pp. 1588
Author(s):  
Valeria Caponetti ◽  
Alexandra Mavridi-Printezi ◽  
Matteo Cingolani ◽  
Enrico Rampazzo ◽  
Damiano Genovese ◽  
...  
Keyword(s):  
Laser Scanning ◽  
Direct Detection ◽  
Low Cost ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Wide Field ◽  
Fluorescence Intensity Ratio ◽  
Green Fluorescence ◽  
One Pot ◽  
Emerging Pollutant

Microplastics (MP) are micrometric plastic particles present in drinking water, food and the environment that constitute an emerging pollutant and pose a menace to human health. Novel methods for the fast detection of these new contaminants are needed. Fluorescence-based detection exploits the use of specific probes to label the MP particles. This method can be environmentally friendly, low-cost, easily scalable but also very sensitive and specific. Here, we present the synthesis and application of a new probe based on perylene-diimide (PDI), which can be prepared in a few minutes by a one-pot reaction using a conventional microwave oven and can be used for the direct detection of MP in water without any further treatment of the sample. The green fluorescence is strongly quenched in water at neutral pH because of the formation dimers. The ability of the probe to label MP was tested for polyvinyl chloride (PVC), polyethylene (PE), polyethylene terephthalate (PET), polypropylene (PP), polystyrene (PS), poly methyl methacrylate (PMMA) and polytetrafluoroethylene (PTFE). The probe showed considerable selectivity to PVC MP, which presented an intense red emission after staining. Interestingly, the fluorescence of the MP after labeling could be detected, under excitation with a blue diode, with a conventional CMOS color camera. Good selectivity was achieved analyzing the red to green fluorescence intensity ratio. UV–Vis absorption, steady-state and time-resolved fluorescence spectroscopy, fluorescence anisotropy, fluorescence wide-field and confocal laser scanning microscopy allowed elucidating the mechanism of the staining in detail.

Cytological observation of the infection process of Venturia carpophila on peach leaves

Plant Disease ◽  
2021 ◽  
Author(s):  
Yang Zhou ◽  
Lei Zhang ◽  
Chuan-Ya Ji ◽  
Chingchai Chaisiri ◽  
Liangfen Yin ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Cell Walls ◽  
Laser Scanning ◽  
Infection Process ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Post Inoculation ◽  
Transmission Electron ◽  
Leaf Surfaces ◽  
Germ Tubes

Peach scab caused by Venturia carpophila, is one of the most destructive fungal diseases of peach worldwide, which seriously affects the peach production. Up to date, the infection process and pathogenesis of V. carpophila on peach remain unclear. Here, we present the infection behaviour of V. carpophila at the ultrastructural and cytological levels in peach leaves with combined microscopic investigations (e.g., light microscopy, confocal laser scanning microscopy, scanning electron microscopy and transmission electron microscopy). V. carpophila germinated at the tip of conidia and produced short germ tubes on peach leaf surfaces at 2 days post-inoculation (dpi). At 3 dpi, swollen tips of germ tubes differentiated into appressoria. At 5 dpi, penetration pegs produced by appressoria broke through the cuticle layer, and then differentiated into thick sub-cuticular hyphae in the pectin layer of the epidermal cell walls. At 10 dpi, the sub-cuticular hyphae extensively colonized in the pectin layer. The primary hyphae ramified into secondary hyphae and proliferated along with the incubation. At 15 dpi, the sub-cuticular hyphae divided laterally to form stromata between the cuticle layer and the cellulose layer of the epidermal cells. At 30 dpi, conidiophores developed from the sub-cuticular stromata. Finally, abundant conidiophores and new conidia appeared on leaf surfaces at 40 dpi. These results provide useful information for further understanding the V. carpophila pathogenesis.

Contraction of Collagen Lattices by Cells from Dupuytren’s Nodules

1996 ◽  
Vol 21 (6) ◽  
pp. 801-805 ◽  
Author(s):  
E. TARPILA ◽  
M. R. GHASSEMIFAR ◽  
S. WINGREN ◽  
M. ÅGREN ◽  
L. FRANZÉN
Keyword(s):  
Laser Scanning ◽  
Cell Types ◽  
Dermal Fibroblasts ◽  
Laser Scanning Microscopy ◽  
Platelet Derived Growth Factor ◽  
Dupuytren’S Disease ◽  
Confocal Laser ◽  
Dupuytren's Disease ◽  
Dermal Cells ◽  
Collagen Lattices

The aim of this study was to see if nodular cells in Dupuytren’s disease differed from dermal cells in their contractile capacity and motility. Ten surgical specimens from patients with Dupuytren’s disease and contracture of the finger of more than 45° were harvested and the nodular cells were explanted and cultured. Dermal fibroblasts from the forearm were used as control cells. Both types of cell had the same growth pattern. The morphology on confocal laser scanning microscopy was also similar in both types of cell. Dermal control cells caused significantly more contraction of collagen lattices compared with fibroblasts from nodules of Dupuytren’s contracture. The F-actin content was equal in both groups. Platelet derived growth factor, PDGF-BB (but not PDGF-AA), increased the chemotactic activity of both cell types, but there were no differences between them. The results indicate that at a late state of the disease cells from Dupuytren’s nodules lose their contractile capacity and regain a phenotype resembling that of dermal fibroblasts.

Sign in / Sign up

Export Citation Format

Share Document