A New Technique for Retarding Fading of Fluorescence: Dpx-Bme

Franklin, A. L.; Filion, W. Gary

doi:10.3109/10520298509113903

Cited by 19 publications

(6 citation statements)

References 19 publications

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“…After fixation, worms were stained with Langeron’s Carmine as previously described [ 35 ]. Worms were then mounted on glass microscope slides in DPX (distyrene, plasticiser, xylene) as previously described [ 36 ] and observed using a Leica TCS SP5II laser scanning confocal microscope, equipped with a 40X oil immersion objective and a 488 nm Argon laser and a 561nm DPSS laser.…”

Section: Methodsmentioning

confidence: 99%

The Diterpenoid 7-Keto-Sempervirol, Derived from Lycium chinense, Displays Anthelmintic Activity against both Schistosoma mansoni and Fasciola hepatica

et al. 2015

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BackgroundTwo platyhelminths of biomedical and commercial significance are Schistosoma mansoni (blood fluke) and Fasciola hepatica (liver fluke). These related trematodes are responsible for the chronic neglected tropical diseases schistosomiasis and fascioliasis, respectively. As no vaccine is currently available for anti-flukicidal immunoprophylaxis, current treatment is mediated by mono-chemical chemotherapy in the form of mass drug administration (MDA) (praziquantel for schistosomiasis) or drenching (triclabendazole for fascioliasis) programmes. This overreliance on single chemotherapeutic classes has dramatically limited the number of novel chemical entities entering anthelmintic drug discovery pipelines, raising significant concerns for the future of sustainable blood and liver fluke control.Methodology/ Principle FindingsHere we demonstrate that 7-keto-sempervirol, a diterpenoid isolated from Lycium chinense, has dual anthelmintic activity against related S. mansoni and F. hepatica trematodes. Using a microtiter plate-based helminth fluorescent bioassay (HFB), this activity is specific (Therapeutic index = 4.2, when compared to HepG2 cell lines) and moderately potent (LD50 = 19.1 μM) against S. mansoni schistosomula cultured in vitro. This anti-schistosomula effect translates into activity against both adult male and female schistosomes cultured in vitro where 7-keto-sempervirol negatively affects motility/behaviour, surface architecture (inducing tegumental holes, tubercle swelling and spine loss/shortening), oviposition rates and egg morphology. As assessed by the HFB and microscopic phenotypic scoring matrices, 7-keto-sempervirol also effectively kills in vitro cultured F. hepatica newly excysted juveniles (NEJs, LD50 = 17.7 μM). Scanning electron microscopy (SEM) evaluation of adult F. hepatica liver flukes co-cultured in vitro with 7-keto-sempervirol additionally demonstrates phenotypic abnormalities including breaches in tegumental integrity and spine loss.Conclusions/ Significance7-keto-sempervirol negatively affects the viability and phenotype of two related pathogenic trematodes responsible for significant human and animal infectious diseases. This plant-derived, natural product is also active against both larval and adult developmental forms. As such, the data collectively indicate that 7-keto-sempervirol is an important starting point for anthelmintic drug development. Medicinal chemistry optimisation of more potent 7-keto-sempervirol analogues could lead to the identification of novel chemical entities useful for future combinatorial or replacement anthelmintic control.

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Section: Methodsmentioning

confidence: 99%

The Diterpenoid 7-Keto-Sempervirol, Derived from Lycium chinense, Displays Anthelmintic Activity against both Schistosoma mansoni and Fasciola hepatica

et al. 2015

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“…This phenomenon, called photobleaching, occurs in confocal microscopy due to the high intensity of the laser illumination and the formation of oxygen radicals as a by‐product of the photochemistry of fluorescence (reaction of oxygen radicals with the fluorescent dyes destroys the dyes). To remedy the photobleaching problem, one can use anti‐fade agents (Franklin & Filion, 1985), such as N ‐propyl gallate, p ‐phenylenediamene (PPD), sodium azide, DABCO, or Citifluor, or apply image enhancement techniques such as contrast manipulation and histogram equalization (Stark & Fitzgerald, 1996).…”

Section: Methodsmentioning

confidence: 99%

Three‐dimensional volume reconstruction of extracellular matrix proteins in uveal melanoma from fluorescent confocal laser scanning microscope images

Bajcsy

Lee

et al. 2006

Journal of Microscopy

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SummaryThe distribution of looping patterns of laminin in uveal melanomas and other tumours has been associated with adverse outcome. Moreover, these patterns are generated by highly invasive tumour cells through the process of vasculogenic mimicry and are not therefore blood vessels. Nevertheless, these extravascular matrix patterns conduct plasma. The threedimensional (3D) configuration of these laminin-rich patterns compared with blood vessels has been the subject of speculation and intensive investigation. We have developed a method for the 3D reconstruction of volume for these extravascular matrix proteins from serial paraffin sections cut at 4 µm thicknesses and stained with a fluorescently labelled antibody to laminin (Maniotis et al., 2002). Each section was examined via confocal laser-scanning focal microscopy (CLSM) and 13 images were recorded in the Z-dimension for each slide. The input CLSM imagery is composed of a set of 3D subvolumes (stacks of 2D images) acquired at multiple confocal depths, from a sequence of consecutive slides. Steps for automated reconstruction included (1) unsupervised methods for selecting an image frame from a subvolume based on entropy and contrast criteria, (2) a fully automated registration technique for image alignment and (3) an improved histogram equalization method that compensates for spatially varying image intensities in CLSM imagery due to photo-bleaching. We compared image alignment accuracy of a fully automated method with registration accuracy achieved by human subjects using a manual method. Automated 3D volume reconstruction was found to provide significant improvement in accuracy, consistency of results and performance time for CLSM images acquired from serial paraffin sections.

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“…There are several known factors that cause spatial intensity heterogeneity, such as (a) photo‐bleaching, (b) fluorescent attenuation along confocal (depth) axis, (c) image acquisition factors (Benson et al ., 1985; Franklin & Filion, 1985; Rigaut & Vassy, 1991), (d) variations of illumination exposure rate, spatially uneven distribution of dye and the spatial characteristics of illumination beams (Oostveldt et al ., 1998) and (e) fluorochrome microenvironment, for example pH, temperature, embedding medium, etc. (Tauer & Hils, 2000).…”

Section: Introductionmentioning

confidence: 99%

Intensity correction of fluorescent confocal laser scanning microscope images by mean‐weight filtering

Lee

Bajcsy

2006

Journal of Microscopy

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SummaryThis paper addresses the problem of intensity correction of fluorescent confocal laser scanning microscope images. Confocal laser scanning microscope images are frequently used in medicine for obtaining 3D information about specimen structures by imaging a set of 2D cross sections and performing 3D volume reconstruction afterwards. However, 2D images acquired from fluorescent confocal laser scanning microscope images demonstrate significant intensity heterogeneity, for example, due to photo-bleaching and fluorescent attenuation in depth. We developed an intensity heterogeneity correction technique that (a) adjusts the intensity heterogeneity of 2D images, (b) preserves fine structural details and (c) enhances image contrast, by performing spatially adaptive mean-weight filtering. Our solution is obtained by formulating an optimization problem, followed by filter design and automated selection of filtering parameters. The proposed filtering method is experimentally compared with several existing techniques by using four quality metrics: contrast, intensity heterogeneity (entropy) in a low frequency domain, intensity distortion in a high frequency domain and saturation. Based on our experiments and the four quality metrics, the developed meanweight filtering outperforms other intensity correction methods by at least a factor of 1.5 when applied to fluorescent confocal laser scanning microscope images.

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A New Technique for Retarding Fading of Fluorescence: Dpx-Bme

Cited by 19 publications

References 19 publications

The Diterpenoid 7-Keto-Sempervirol, Derived from Lycium chinense, Displays Anthelmintic Activity against both Schistosoma mansoni and Fasciola hepatica

The Diterpenoid 7-Keto-Sempervirol, Derived from Lycium chinense, Displays Anthelmintic Activity against both Schistosoma mansoni and Fasciola hepatica

Three‐dimensional volume reconstruction of extracellular matrix proteins in uveal melanoma from fluorescent confocal laser scanning microscope images

Intensity correction of fluorescent confocal laser scanning microscope images by mean‐weight filtering

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