Development of high-throughput methods to quantify cysts of Toxoplasma gondii
Delphine AldebertMagali HypolitePierre CavaillèsBastien TouquetPierre FloriCorinne LoeuilletM.-F. Cesbron-Delauw
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Abstract Toxplasma is a protozoan parasite, which forms persistent cysts in tissues of chronically infected animals and humans. Cysts can reactivate leading to severe pathologies. They also contribute to the transmission of Toxoplasma infection in humans by ingestion of undercooked meat. Classically, the quantification of cyst burden in tissues uses microscopy methods, which are laborious and time consuming. Here, we have developed automated protocols to quantify cysts, based on flow cytometry or high‐throughput microscopy. Brains of rodents infected with cysts of Prugniaud strain were incubated with the FITC‐ Dolichos biflorus lectin and analyzed by flow cytometry and high‐throughput epifluorescence microscopy. The comparison of cyst counts by manual epifluorescence microscopy to flow cytometry or to high‐throughput epifluorescence microscopy revealed a good correlation ( r = 0.934, r = 0.993, P < 0.001 respectively). High‐throughput epifluorescence microscopy was found to be more specific and sensitive than flow cytometry and easier to use for large series of samples. This reliable and easy protocol allow the specific detection of Toxoplasma cysts in brain, even at low concentrations; it could be a new way to detect them in water and in contaminate food. © 2011 International Society for Advancement of CytometryKeywords:
Cytometry
The discrimination of out of focus contributions in fluorescence microscopy possible in a confocal setup will establish itself as a supplement to conventional fluorescence microscopy. The improvement of the contrast compared with conventional fluorescence microscopy depends mainly on the density of the fluorescing material and the thickness of the sample. The term thickness, that which microscopists refer to as the size of the specimen along the optical axis, will gain a new quality since a confocal fluorescence microscope may reveal totally different features when recording data in planes that are 0.3μm apart. Differences that have in the past been neglected suddenly become important. The following article will outline important features in the application of confocal fluorescence microscopy in the biological sciences, point out its limitatk'ns, and draw attention to expected developments.
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This study was carried out to evaluate the anti-parasitic effect of ursolic acid against Toxoplasma gondii (T. gondii) that induces toxoplasmosis, particularly in humans. The anti-parasitic effects of ursolic acid against T. gondii-infected cells and T. gondii were evaluated through different specific assays, including immunofluorescence staining and animal testing. Ursolic acid effectively inhibited the proliferation of T. gondii when compared with sulfadiazine, and consistently induced anti-T. gondii activity/effect. In particular, the formation of parasitophorous vacuole membrane (PVM) in host cells was markedly decreased after treating ursolic acid, which was effectively suppressed. Moreover, the survival rate of T. gondii was strongly inhibited in T. gondii group treated with ursolic acid, and then 50% inhibitory concentration (IC50) against T. gondii was measured as 94.62 μg/mL. The T. gondii-infected mice treated with ursolic acid indicated the same survival rates and activity as the normal group. These results demonstrate that ursolic acid causes anti-T. gondii action and effect by strongly blocking the proliferation of T. gondii through the direct and the selective T. gondii-inhibitory ability as well as increases the survival of T. gondii-infected mice. This study shows that ursolic acid has the potential to be used as a promising anti-T. gondii candidate substance for developing effective anti-parasitic drugs.
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Journal Article Confocal Scanning Fluorescence Microscopy: A New Method for Phagocytosis Research Get access Gregory R Hook, Gregory R Hook Casualty Care Research Department. Pathophysiology Division, Naval Medical Research Institute, Bethesda, Maryland Reprint requests: Gregory Hook. Naval Medical Research Institute. Bldg. 17. MS 10, NMCNCR, Bethesda, MD 20814. Search for other works by this author on: Oxford Academic Google Scholar Charles O Odeyale Charles O Odeyale Casualty Care Research Department. Pathophysiology Division, Naval Medical Research Institute, Bethesda, Maryland Search for other works by this author on: Oxford Academic Google Scholar Journal of Leukocyte Biology, Volume 45, Issue 4, April 1989, Pages 277–282, https://doi.org/10.1002/jlb.45.4.277 Published: 01 April 1989 Article history Received: 25 August 1988 Accepted: 24 October 1988 Published: 01 April 1989
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Image cytometry is mostly associated with high quality but low quantity, whereas flow cytometry is associated with high quantity but low quality. Achieving both quality and quantity in cytometry is possible in the case of automated confocal microscopy systems. We have shown that the automation of image acquisition and image processing in confocal microscopy is possible for specific tasks such as 2D and 3D FISH imaging. This paper is focused on practical problems and solutions that we have encountered during our research. We believe that our experience might be helpful to all those who decide to automate their light microscopy systems for cell studies, i.e. for image cytometry purposes.
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Fluorescence-lifetime imaging microscopy
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Early response of cancer cells to chemical compounds and chemotherapeutic drugs were studied using novel fluorescence tools and microscopy techniques. We applied confocal microscopy, two-photon fluorescence lifetime imaging microscopy and super-resolution localization-based microscopy to assess structural and functional changes in cancer cells in vitro. The dynamics of energy metabolism, intracellular pH, caspase-3 activation during staurosporine-induced apoptosis as well as actin cytoskeleton rearrangements under chemotherapy were evaluated. We have showed that new genetically encoded sensors and advanced fluorescence microscopy methods provide an efficient way for multiparameter analysis of cell activities
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Two-photon excitation microscopy
Fluorescence-lifetime imaging microscopy
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Objective To seek a simple proper preservation method of Toxoplasma gondii strains and provide foundation for further study.Methods Cryopreservation of Toxoplasma gondii strains by using refrigerator in different temperature and cell culture conservation were tested.Results Toxoplasma gondii can be preserved at(-196°C,)(-70°C) permanently,and(4°C) for 14 days.Plenty of Toxoplasma gondii can be obtained by cell culture and it can infect almost every kinds of cells except erythroplastid.Conclusion The virulence of Toxoplasma gondii was steady after two preservation methods.
Toxoplasmosis
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Confocal fluorescence microscopy is particularly well-known from the beautiful
images that have been obtained with this technique from cells. Several cellular
components could be nicely visualized simultaneously by staining them with
different fluorophores. Not only for ensemble applications but also in single
molecule research confocal fluorescence microscopy became a popular technique.
In this thesis the possibilities are shown to study a complicated biological process, which is Nucleotide Excision Repair (NER), on the single molecule level with confocal fluorescence microscopy. In chapter one an introduction is given in single molecule fluorescence, properties of fluorescent labels and immobilization methods. Also the NER process is described and the configuration of the confocal fluorescence microscope used.
Fluorescence-lifetime imaging microscopy
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