Semiquantitative Confocal Laser Scanning Microscopy Applied to Marine Invertebrate Ecotoxicology

Chandler, G. Thomas; Volz, David C.

doi:10.1007/s10126-002-0105-y

Cited by 8 publications

(6 citation statements)

References 19 publications

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“…This nondestructive method allows the qualitative detection of ingestion in whole animals and the determination of the area of accumulation. Confocal microscopy also allows noninvasive optical sectioning [34] because illumination, specimen, and detector have the same focus [35]. The final image corresponds to the point of focus in the specimen.…”

Section: Introductionmentioning

confidence: 99%

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A comparison of nanoparticle and fine particle uptake by Daphnia magna

Rosenkranz

Chaudhry²,

Stone

et al. 2009

Enviro Toxic and Chemistry

298

219

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The use of nanoparticles in various applications is steadily on the rise, with use in a range of applications, including printer toner, sunscreen, medical imaging, and enhanced drug delivery. While research on human effects via, for example, inhalation is relatively well developed, the environmental assessment of nanoparticles is in its infancy. In the present study, we assessed the uptake and quantitative accumulation, as well as the depuration, of a model nanoparticle, a 20-nm fluorescent carboxylated polystyrene bead, in the aquatic invertebrate Daphnia magna and compared it to a larger, 1,000-nm particle. Using confocal microscopy, rapid accumulation in the gastrointestinal tract was observed within an hour of exposure to both particle sizes in both adults and neonates. Fluorescence could also be observed in the oil storage droplets, suggesting that both particle sizes have crossed the gut's epithelial barrier. Quantification of fluorescence of both sizes of particles showed that although uptake of the 20-nm particles was lower in terms of mass it was equal to or greater than 1000-nm particle uptake when expressed as surface area or particle number. Depuration was relatively rapid for the 1000-nm beads, decreasing by more than 90% over 4 h. In contrast, depuration of the 20-nm beads was less extensive, reaching 40% over 4 h. Transmission electron microscopy confirmed uptake of 1,000-nm beads, but uptake of 20-nm beads was inconclusive since similar-sized inclusions could be observed in control treatments.

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Section: Introductionmentioning

confidence: 99%

“…This enables the location of fluorescence, for example, within cell organelles instead of having the uncertainty if it is inside or on the surface of the organelle. A similar method has been used in ecotoxicological studies to assess the survival rate of crustacean eggs [34,35].…”

Section: Introductionmentioning

confidence: 99%

A comparison of nanoparticle and fine particle uptake by Daphnia magna

Rosenkranz

Chaudhry²,

Stone

et al. 2009

Enviro Toxic and Chemistry

298

219

View full text Add to dashboard Cite

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“…The calculated linear fit indicates that there is additional BODIPY fluorescence (1682 FIU) which is not accounted for by the TAGs measured via liquid-state 1 H NMR. We hypothesize that this is due to fluorescence from BODIPY molecules accumulated in non-TAG lipids such as phospholipids [ 28 , 50 ]. Liquid-state NMR only samples protons in molecules that can rotate freely, such as TAGs present within cytoplasmic lipid deposits [ 4 ].…”

Section: Resultsmentioning

confidence: 99%

Measurement of Lipid Accumulation in Chlorella vulgaris via Flow Cytometry and Liquid-State ¹H NMR Spectroscopy for Development of an NMR-Traceable Flow Cytometry Protocol

et al. 2015

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In this study, we cultured Chlorella vulgaris cells with a range of lipid contents, induced via nitrogen starvation, and characterized them via flow cytometry, with BODIPY 505/515 as a fluorescent lipid label, and liquid-state 1H NMR spectroscopy. In doing so, we demonstrate the utility of calibrating flow cytometric measurements of algal lipid content using triacylglyceride (TAG, also known as triacylglycerol or triglyceride) content per cell as measured via quantitative 1H NMR. Ensemble-averaged fluorescence of BODIPY-labeled cells was highly correlated with average TAG content per cell measured by bulk NMR, with a linear regression yielding a linear fit with r 2 = 0.9974. This correlation compares favorably to previous calibrations of flow cytometry protocols to lipid content measured via extraction, and calibration by NMR avoids the time and complexity that is generally required for lipid quantitation via extraction. Flow cytometry calibrated to a direct measurement of TAG content can be used to investigate the distribution of lipid contents for cells within a culture. Our flow cytometry measurements showed that Chlorella vulgaris cells subjected to nitrogen limitation exhibited higher mean lipid content but a wider distribution of lipid content that overlapped the relatively narrow distribution of lipid content for replete cells, suggesting that nitrogen limitation induces lipid accumulation in only a subset of cells. Calibration of flow cytometry protocols using direct in situ measurement of TAG content via NMR will facilitate rapid development of more precise flow cytometry protocols, enabling investigation of algal lipid accumulation for development of more productive algal biofuel feedstocks and cultivation protocols.

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“…Currently, morphological studies in Actiniaria combine complementary techniques: gross dissections provide information on internal features in three dimensions with low resolution whereas histology provides resolution at the cellular level in two dimensions. Hence, micro-CT methods combine qualities of both methods (i.e., high resolution with tridimensional qualities) with comparable cost (Chandler and Volz, 2004) while avoiding some of their shortcomings (e.g., slowness, sample preparation, small volumes, artifacts) (Shearer et al, 2016). When compared to dissections and histology, micro-CT scanning is also presumed to be nondestructive, easy, and fast.…”

Section: Fig 12 (Continued) Diadumene Lineatamentioning

confidence: 99%

Sea Anemones through X-Rays: Visualization of Two Species ofDiadumene(Cnidaria, Actiniaria) Using Micro-CT

Gusmão

Grajales

Rodríguez

2018

American Museum Novitates

821

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Diadumenidae is a monogeneric family comprised of 10 valid species in the genus Diadumene. Although these species are distributed in all but the Southern Ocean, Diadumene lineata is a cosmopolitan species and one of the most widely distributed marine organisms. Recent phylogenetic analyses with multiple species have recovered Diadumenidae as a monophyletic group with high support. Although generic placement is straightforward, species-level identifications within Diadumene are difficult because species are defined by a mosaic of characters that vary in degree rather than kind. Two species of the genus have been recorded in the southwestern Atlantic: D. paranaensis along the south coast of Brazil and D. lineata along the southeast and northeast coasts of Brazil as well as in Argentina. Here we record D. leucolena for the first time in the southern hemisphere and describe D. manezinha, sp. nov., raising to four the number of species of Diadumene for Brazil and to 11 worldwide. We incorporated microcomputed tomography (micro-CT) into the description of both species in the first application of the method for Actiniaria. We evaluate the utility of micro-CT imaging and its potential to generate fast, low-cost, and high-resolution datasets despite the anemone's low-density soft tissue. We present a protocol for sample handling, chemical staining, and scanning parameters that resulted in satisfactory imaging of the two specimens examined. We also analyze advantages and limitations of using micro-CT over traditional techniques in the study of sea anemones.

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Semiquantitative Confocal Laser Scanning Microscopy Applied to Marine Invertebrate Ecotoxicology

Cited by 8 publications

References 19 publications

A comparison of nanoparticle and fine particle uptake by Daphnia magna

A comparison of nanoparticle and fine particle uptake by Daphnia magna

Measurement of Lipid Accumulation in Chlorella vulgaris via Flow Cytometry and Liquid-State ¹H NMR Spectroscopy for Development of an NMR-Traceable Flow Cytometry Protocol

Sea Anemones through X-Rays: Visualization of Two Species ofDiadumene(Cnidaria, Actiniaria) Using Micro-CT

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