Darkfield-Confocal Microscopy detection of nanoscale particle internalization by human lung cells

<p>Abstract</p> <p>Background</p> <p>Concerns over the health effects of nanomaterials in the environment have created a need for microscopy methods capable of examining the biological interactions of nanoparticles (NP). Unfortunately, NP are beyond the diffraction limit of resolution for conventional light microscopy (~200 nm). Fluorescence and electron microscopy techniques commonly used to examine NP interactions with biological substrates have drawbacks that limit their usefulness in toxicological investigation of NP. EM is labor intensive and slow, while fluorescence carries the risk of photobleaching the sample and has size resolution limits. In addition, many relevant particles lack intrinsic fluorescence and therefore can not be detected in this manner. To surmount these limitations, we evaluated the potential of a novel combination of darkfield and confocal laser scanning microscopy (DF-CLSM) for the efficient 3D detection of NP in human lung cells. The DF-CLSM approach utilizes the contrast enhancements of darkfield microscopy to detect objects below the diffraction limit of 200 nm based on their light scattering properties and interfaces it with the power of confocal microscopy to resolve objects in the z-plane.</p> <p>Results</p> <p>Validation of the DF-CLSM method using fluorescent polystyrene beads demonstrated spatial colocalization of particle fluorescence (Confocal) and scattered transmitted light (Darkfield) along the X, Y, and Z axes. DF-CLSM imaging was able to detect and provide reasonable spatial locations of 27 nm TiO₂particles in relation to the stained nuclei of exposed BEAS 2B cells. Statistical analysis of particle proximity to cellular nuclei determined a significant difference between 5 min and 2 hr particle exposures suggesting a time-dependant internalization process.</p> <p>Conclusions</p> <p>DF-CLSM microscopy is an alternative to current conventional light and electron microscopy methods that does not rely on particle fluorescence or contrast in electron density. DF-CLSM is especially well suited to the task of establishing the spatial localization of nanoparticles within cells, a critical topic in nanotoxicology. This technique has advantages to 2D darkfield microscopy as it visualizes nanoparticles in 3D using confocal microscopy. Use of this technique should aid toxicological studies related to observation of NP interactions with biological endpoints at cellular and subcellular levels.</p

Better Together: Unifying Datalog and Equality Saturation

We present egglog, a fixpoint reasoning system that unifies Datalog and equality saturation (EqSat). Like Datalog, it supports efficient incremental execution, cooperating analyses, and lattice-based reasoning. Like EqSat, it supports term rewriting, efficient congruence closure, and extraction of optimized terms. We identify two recent applications--a unification-based pointer analysis in Datalog and an EqSat-based floating-point term rewriter--that have been hampered by features missing from Datalog but found in EqSat or vice-versa. We evaluate egglog by reimplementing those projects in egglog. The resulting systems in egglog are faster, simpler, and fix bugs found in the original systems.Comment: PLDI 202

An Integrated Imaging Approach to the Study of Oxidative Stress Generation by Mitochondrial Dysfunction in Living Cells

BACKGROUND: The mechanisms of action of many environmental agents commonly involve oxidative stress resulting from mitochondrial dysfunction. Zinc is a common environmental metallic contaminant that has been implicated in a variety of oxidant-dependent toxicological responses. Unlike ions of other transition metals such as iron, copper, and vanadium, Zn(2+) does not generate reactive oxygen species (ROS) through redox cycling. OBJECTIVE: To characterize the role of oxidative stress in zinc-induced toxicity. METHODS: We used an integrated imaging approach that employs the hydrogen peroxide (H2O2)-specific fluorophore Peroxy Green 1 (PG1), the mitochondrial potential sensor 5,5 ,6,6 -tetrachloro-1,1 ,3,3 -tetraethylbenzimidazolylcarbocyanine iodide (JC-1), and the mitochondria-targeted form of the redox-sensitive genetically encoded fluorophore MTroGFP1 in living cells. RESULTS: Zinc treatment in the presence of the Zn(2+) ionophore pyrithione of A431 skin carcinoma cells preloaded with the H(2)O(2)-specific indicator PG1 resulted in a significant increase in H(2)O(2) production that could be significantly inhibited with the mitochondrial inhibitor carbonyl cyanide 3-chlorophenylhydrazone. Mitochondria were further implicated as the source of zinc-induced H(2)O(2) formation by the observation that exposure to zinc caused a loss of mitochondrial membrane potential. Using MTroGFP1, we showed that zinc exposure of A431 cells induces a rapid loss of reducing redox potential in mitochondria. We also demonstrated that zinc exposure results in rapid swelling of mitochondria isolated from mouse hearts. CONCLUSION: Taken together, these findings show a disruption of mitochondrial integrity, H(2)O(2) formation, and a shift toward positive redox potential in cells exposed to zinc. These data demonstrate the utility of real-time, live-cell imaging to study the role of oxidative stress in toxicological responses

Predictions for the frequency and orbital radii of massive extrasolar planets

We investigate the migration of massive extrasolar planets due to gravitational interaction with a viscous protoplanetary disc. We show that a model in which planets form at 5 AU at a constant rate, before migrating, leads to a predicted distribution of planets that is a steeply rising function of log (a), where a is the orbital radius. Between 1 AU and 3 AU, the expected number of planets per logarithmic interval in orbital radius roughly doubles. We demonstrate that, once selection effects are accounted for, this is consistent with current data, and then extrapolate the observed planet fraction to masses and radii that are inaccessible to current observations. In total, about 15 percent of stars targeted by existing radial velocity searches are predicted to possess planets with masses 0.3 M_Jupiter < M_p sin (i) < 10 M_Jupiter, and radii 0.1 AU < a < 5 AU. A third of these planets (around 5 percent of the target stars) lie at the radii most amenable to detection via microlensing. A further 5-10 percent of stars could have planets at radii of 5 AU < a < 8 AU that have migrated outwards. We discuss the probability of forming a system (akin to the Solar System) in which significant radial migration of the most massive planet does not occur. About 10-15 percent of systems with a surviving massive planet are estimated to fall into this class. Finally, we note that a smaller fraction of low mass planets than high mass planets is expected to survive without being consumed by the star. The initial mass function for planets is thus predicted to rise more steeply towards small masses than the observed mass function.Comment: MNRAS, in pres