scholarly journals Silencing cuticular pigmentation genes enables RNA FISH in intact chemosensory appendagess

2018 ◽  
Author(s):  
Stefan Pentzold ◽  
Veit Grabe ◽  
Andrei Ogonkov ◽  
Lydia Schmidt ◽  
Wilhelm Boland ◽  
...  
Keyword(s):  
Laser Scanning ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Microscopy Imaging ◽  
Leaf Beetles ◽  
Antisense Mrna ◽  
Rna Fish ◽  
Whole Mount ◽  
Light Absorbance ◽  
Odorant Binding

AbstractOptical imaging of gene expression by RNA-fluorescent in situ hybridisation (FISH) in whole-mount sensory appendages of insects is often impeded by their highly pigmented cuticle. Since most chemical bleaching agents are incompatible with imaging fluorescent-labelled nucleotides, we developed a RNA interference-based method for clearing cuticular pigmentation that allows imaging of fluorescent mRNA in whole-mount appendages of insects. Silencing key genes of the tyrosine-derived pigmentation pathway by injecting dsRNA of laccase2 or tyrosine hydroxylase in two leaf beetles species (Chrysomela populi, Phaedon cochleariae) resulted in clearance of the highly pigmented cuticle and in significant decreased light absorbance. Intact chemosensory appendages (palps, antennae and legs) from RNAi-cleared individuals were used to image expression and spatial distribution of antisense mRNA of two chemosensory genes (gustatory receptor, odorant-binding protein) via RNA FISH and confocal laser scanning microscopy. Imaging of these genes did neither work for RNAi-controls (dsGfp) due to retained pigmentation, nor for FISH-controls using sense mRNA. Furthermore, we show that several chemical bleaching agents are not feasible with FISH, either due to significant degradation of polynucleotides, lack of clearing efficacy or long incubation times. Overall, silencing pigmentation genes is a significant improvement over bleaching agents allowing fluorescence imaging in whole-mount appendages and organs.

scholarly journals Use of electromagnetic stimulation on an Enterococcus faecalis biofilm on root canal treated teeth in vitro

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Beatriz H. D. Panariello ◽  
Justin K. Kindler ◽  
Kenneth J. Spolnik ◽  
Ygal Ehrlich ◽  
George J. Eckert ◽  
...  
Keyword(s):  
Enterococcus Faecalis ◽  
Root Canal ◽  
Laser Scanning ◽  
Confocal Imaging ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Microscopy Imaging ◽  
Electromagnetic Stimulation ◽  
Root Canal Disinfection

AbstractRoot canal disinfection is of utmost importance in the success of the treatment, thus, a novel method for achieving root canal disinfection by electromagnetic waves, creating a synergistic reaction via electric and thermal energy, was created. To study electromagnetic stimulation (EMS) for the disinfection of root canal in vitro, single rooted teeth were instrumented with a 45.05 Wave One Gold reciprocating file. Specimens were sterilized and inoculated with Enterococcus faecalis ATCC 29,212, which grew for 15 days to form an established biofilm. Samples were treated with 6% sodium hypochlorite (NaOCl), 1.5% NaOCl 1.5% NaOCl with EMS, 0.9% saline with EMS or 0.9% saline. After treatments, the colony forming units (CFU) was determined. Data was analyzed by Wilcoxon Rank Sums Test (α = 0.05). One sample per group was scored and split for confocal laser scanning microscopy imaging. There was a significant effect with the use of NaOCl with or without EMS versus 0.9% saline with or without EMS (p = 0.012 and 0.003, respectively). CFUs were lower when using 0.9% saline with EMS versus 0.9% saline alone (p = 0.002). Confocal imaging confirmed CFU findings. EMS with saline has an antibiofilm effect against E. faecalis and can potentially be applied for endodontic disinfection.

GABA-induced increases in [Ca2+]i in retinal neurons of postnatal rabbits

1996 ◽  
Vol 13 (3) ◽  
pp. 441-447 ◽  
Author(s):  
Bo Huang ◽  
Dianna A. Redburn
Keyword(s):  
Gaba Receptor ◽  
Laser Scanning ◽  
Synapse Formation ◽  
Plexiform Layer ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Retinal Neurons ◽  
Retinal Cells ◽  
Whole Mount ◽  
Trophic Role

AbstractPrevious studies have indicated that γ-aminobutyric acid (GABA) plays an important trophic role in the synapse formation between horizontal cells and photoreceptors in postnatal rabbit retina. However, the mechanism of the GABA effect has not been identified. Using fluo-3 Ca2+ imaging and confocal laser scanning microscopy we examined the effect of GABA on [Ca2+]i during postnatal retinal development. GABA (100 μM) evoked a fast and transient increase of [Ca2+]i in selected populations of freshly dissociated retinal cells from postnatal rabbits. This increase was apparent on postnatal day 1 and reached a maximum on day 5. Little increase in [Ca2+]i, was observed in retinal cells isolated from adult rabbits. GABA receptor antagonists, picrotoxin and bicuculline, significantly reduced the response. The GABAU agonist, baclofen, did not evoke any [Ca2+]i changes. The GABA-induced increase in [Ca2+]i, was observed in all retinal layers in neonatal retinal whole-mount explants. In the outer retina, the increase was seen in cone photoreceptors which were specifically labeled with peanut agglutinin (PNA). The GABA-induced increase in [Ca2+]i may provide an important mechanism for regulating cone synaptogenesis in the outer plexiform layer of the postnatal retina.

Automated biofilm morphology quantification from confocal laser scanning microscopy imaging

2003 ◽  
Vol 47 (5) ◽  
pp. 31-37 ◽  
Author(s):  
J.B. Xavier ◽  
D.C. White ◽  
J.S. Almeida
Keyword(s):  
Confocal Laser Scanning Microscopy ◽  
Laser Scanning ◽  
Laser Scanning Microscopy ◽  
Confocal Laser Scanning ◽  
Scanning Microscopy ◽  
Confocal Laser ◽  
Microscopy Imaging ◽  
Biofilm Structure ◽  
User Friendly ◽  
Processing Steps

In spite of the immediate visual appeal of confocal laser scanning microscopy images, the extraction of accurate reconstitutions of biofilm morphology requires a lengthy and computational intensive succession of processing steps. However, once performed, it provides ample reward by enabling the quantitative study of biofilm structure. A software suite of image processing tools for full automation of biofilm morphology quantification was developed by integrating preprocessing, segmentation and morphology quantification operations. This software toolbox was implemented in a web server and a user friendly interface was developed to facilitate image submission, storage and sharing, its access being unrestricted for scientific applications. The image bioinformatics tool which results from the integration of the processing operations can be accessed at http://www.itqb.unl.pt:1111/clsmip/. Its use is described in this paper and is illustrated with an example of processing of experimental data describing the growth of a mixed species denitrifying biofilm.

The furfuryl alcohol (FA) resin distribution in the furfurylated bamboo

Holzforschung ◽  
2020 ◽  
Vol 0 (0) ◽  
Author(s):  
Wanju Li ◽  
Minghui Liu ◽  
Hankun Wang ◽  
Hongbo Zhai ◽  
Yan Yu
Keyword(s):  
Furfuryl Alcohol ◽  
Laser Scanning ◽  
Parenchymal Cell ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Sem Images ◽  
Microscopy Imaging ◽  
Resin Impregnation ◽  
Specific Distribution ◽  
Ft Ir

AbstractIt is well known the properties of resin impregnation wood is significantly influenced by the specific distribution pattern of resin in the modified wood. In this work, bamboo was furfurylated with an improved process. In addition to testing and evaluating its main physical, mechanical and durable properties, it was explored how the furfuryl alcohol (FA) resin is distributed in the furfurylated bamboo. To achieve this goal, scanning electron microscopy (SEM), confocal laser scanning microscopy (CLSM), nanoindentation, and imaging Fourier transform infrared microscopy (imaging FT-IR) were applied. SEM images demonstrates FA resin is mainly located in the parenchymal cell cavity of bamboo, however the existence of FA resin in the small cavities of the bamboo fibers is also observed by CLSM. In addition, the result of nanoindentation and imaging FT-IR both indicates that FA can penetrate and polymerize within the cell wall of the bamboo fiber. It is then concluded the heterogeneous but multi-scale distribution of FA resin should be responsible for the significant improvement of furfurylated bamboo in both dimensional stability and biological durability.

Assessment of three-dimensional biofilm models through direct comparison with confocal microscopy imaging

2004 ◽  
Vol 49 (11-12) ◽  
pp. 177-185 ◽  
Author(s):  
J.B. Xavier ◽  
C. Picioreanu ◽  
M.C.M. van Loosdrecht
Keyword(s):  
Structure Formation ◽  
Laser Scanning ◽  
Three Dimensional ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Experimental Conditions ◽  
Microscopy Imaging ◽  
Biofilm Growth ◽  
Biofilm Development ◽  
Biofilm Structure

The mathematical modeling of spatial biofilm formation that provides the capability to predict biofilm structure from first principles has been in development for the past six years. However, a direct and quantitative link between model predictions and the experimentally observed structure formation still remains to be established. This work assesses the capability of a state-of-the-art technique for three-dimensional (3D) modeling of biofilm structure, individual based modeling (IbM), to quantitatively describe the early development of a multispecies denitrifying biofilm. Model evaluation was carried out by comparison of predicted structure with that observed from two experimental datasets using confocal laser scanning microscopy (CLSM) monitoring of biofilm development in laboratory flowcells. Experimental conditions provided biofilm growth without substrate limitation, which was confirmed from substrate profiles computed by the model. 3D structures were compared quantitatively using a set of morphological parameters including the biovolume, filled-space profiles, substratum coverage, average thickness and normalized roughness. In spite of the different morphologies detectable in the two independent short-term experiments analyzed here, the model was capable of accurate fitting data from both experiments. Prediction of structure formation was precise, as expressed by the set of morphology parameters used.

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