Time-lapse 3-D measurements of a glucose biosensor in multicellular spheroids by light sheet fluorescence microscopy in commercial 96-well plates

Light sheet fluorescence microscopy has previously been demonstrated on a commercially available inverted fluorescence microscope frame using the method of oblique plane microscopy (OPM). In this paper, OPM is adapted to allow time-lapse 3-D imaging of 3-D biological cultures in commercially available glass-bottomed 96-well plates using a stage-scanning OPM approach (ssOPM). Time-lapse 3-D imaging of multicellular spheroids expressing a glucose Förster resonance energy transfer (FRET) biosensor is demonstrated in 16 fields of view with image acquisition at 10 minute intervals. As a proof-of-principle, the ssOPM system is also used to acquire a dose response curve with the concentration of glucose in the culture medium being varied across 42 wells of a 96-well plate with the whole acquisition taking 9 min. The 3-D image data enable the FRET ratio to be measured as a function of distance from the surface of the spheroid. Overall, the results demonstrate the capability of the OPM system to measure spatio-temporal changes in FRET ratio in 3-D in multicellular spheroids over time in a multi-well plate format

Polarization Diagnostics for Cool Core Cluster Emission Lines

The nature of the interaction between low-excitation gas filaments at ~104 K, seen in optical line emission, and diffuse X-ray emitting coronal gas at ~107 K in the centers of galaxy clusters remains a puzzle. The presence of a strong, empirical correlation between the two gas phases is indicative of a fundamental relationship between them, though as yet of undetermined cause. The cooler filaments, originally thought to have condensed from the hot gas, could also arise from a merger or the disturbance of cool circumnuclear gas by nuclear activity. Here, we have searched for intrinsic line emission polarization in cool core galaxy clusters as a diagnostic of fundamental transport processes. Drawing on developments in solar astrophysics, direct energetic particle impact induced polarization holds the promise to definitively determine the role of collisional processes such as thermal conduction in the ISM physics of galaxy clusters, while providing insight into other highly anisotropic excitation mechanisms such as shocks, intense radiation fields, and suprathermal particles. Under certain physical conditions, theoretical calculations predict of the order of 10% polarization. Our observations of the filaments in four nearby cool core clusters place stringent upper limits ( 0.1%) on the presence of emission line polarization, requiring that if thermal conduction is operative, the thermal gradients are not in the saturated regime. This limit is consistent with theoretical models of the thermal structure of filament interfacesPeer reviewe

Anomalous cooling of the massive white dwarf in U Geminorum following a narrow dwarf nova outburst

We obtained Hubble GHRS medium resolution (G160M grating) phase-resolved spectroscopic observations of the prototype dwarf nova U Geminorum during dwarf nova quiescence, 13 days and 61 days following the end of a narrow outburst. The spectral wavelength ranges were centered upon three different line regions: N V (1238\AA, 1242\AA), Si III (1300\AA) and He II (1640\AA). All of the quiescent spectra at both epochs are dominated by absorption lines and show no emission features. The Si III and He II absorption line velocities versus orbital phase trace the orbital motion of the white dwarf but the N~V absorption velocities appear to deviate from the white dwarf motion. We confirm our previously reported low white dwarf rotational velocity, V sin i= 100 km/s. We obtain a white dwarf orbital velocity semi-amplitude K1=107 km/s. Using the gamma-velocity of Wade (1981) we obtain an Einstein redshift of 80.4 km/s and hence a carbon core white dwarf mass of ~1.1 Msun. We report the first subsolar chemical abundances of C and Si for U Gem with C down by 0.05 with respect to the Sun, almost certainly a result of C depletion due to thermonuclear processing. This C-depletion is discussed within the framework of a weak TNR, contamination of the secondary during the common envelope phase, and mixing of C-depleted white dwarf gas with C-depleted matter deposited during a dwarf nova event. Remarkably the Teff of the white dwarf 13 days after outburst is only 32,000K, anomalously cooler than previous early post-outburst measurements. Extensive cooling during an extraordinarily long (210 days) quiescence followed by accretion onto an out-of-equilibrium cooled degenerate could explain the lower Teff.Comment: 16 pages AAS-Latex, 4 Figures, accepted for publication in Ap

Leadership Integrity and Diversity in the Workplace

Leadership integrity and diversity are significant factors in the relationship between leader and employee in the workplace. For employees to follow their leaders, they want someone that they can trust. They will not follow those whom they do not trust or who will not or cannot disclose a clear set of values, ethics and standards. This research examined 941 responses from workers in the United States who completed the Perceived Leader Integrity Scale (PLIS) developed by Craig and Gustafson (1998) and the Workplace Diversity Survey by De Meuse and Hostager (1996), along with demographic questions. This research also examined the relationship of perceived leadership integrity and workplace diversity

Adaptive light-sheet fluorescence microscopy with a deformable mirror for video-rate volumetric imaging

Light-sheet fluorescence microscopy (LSFM) achieves optically sectioned imaging with the relatively low photobleaching and phototoxic effect. To achieve high-speed volumetric LSFM imaging without perturbing the sample, it is necessary to use some form of remote refocusing in the detection beam path. Previous work used electrically tunable lenses, tunable acoustic gradient index of refraction lenses, or the remote-refocusing approach of Botcherby et al. [Opt. Lett. 32(14), 2007 (2007)] to achieve remote refocusing. However, these approaches generally only provide low-order defocus correction, which is not compatible with higher-NA objectives that require higher order defocus corrections or reduce the optical throughput. In order to simultaneously achieve high-speed remote refocusing and correct system aberrations, we employ a deformable mirror in the detection path that is capable of providing higher orders of defocus and aberration correction in an optical system with an NA of 0.72–0.75. We demonstrate high-speed volumetric imaging at 26.3 volumes per second and 35 frames per volume for a defocus range of −50 to 50 μm

Anomalous behavior of spin wave resonances in Ga_{1-x}Mn_{x}As thin films

We report ferromagnetic and spin wave resonance absorption measurements on high quality epitaxially grown Ga_{1-x}Mn_{x}As thin films. We find that these films exhibit robust ferromagnetic long-range order, based on the fact that up to seven resonances are detected at low temperatures, and the resonance structure survives to temperatures close to the ferromagnetic transition. On the other hand, we observe a spin wave dispersion which is linear in mode number, in qualitative contrast with the quadratic dispersion expected for homogeneous samples. We perform a detailed numerical analysis of the experimental data and provide analytical calculations to demonstrate that such a linear dispersion is incompatible with uniform magnetic parameters. Our theoretical analysis of the ferromagnetic resonance data, combined with the knowledge that strain-induced anisotropy is definitely present in these films, suggests that a spatially dependent magnetic anisotropy is the most likely reason behind the anomalous behavior observed.Comment: 9 pages, including 6 figure

Some factors influencing populations of the European corn borer, Ostrinia nubilalis (Hubner) in the north central states: Resistance of corn, time of planting and weather conditions Part II, 1958-1962

A cooperative project was conducted by the agricultural experiment stations of Iowa, Minnesota and Ohio and the U. S. Department of Agriculture to study the effects of weather, planting date and resistant hybrids as factors influencing populations of the European com borer, Ostrinia nubilalis (Hübner). Identical studies were carried out at Ankeny, Iowa; Waseca, Minnesota; and Wooster, Ohio, during a 10-year period, 1953-1962. The first 4 years of the study (1953-56) were reported by Everett et al. (1958). The work reported herein is a companion bulletin to the Everett et al. (1958) publication and deals with the results of experiments conducted during 1958-1962. The experimental design was a randomized block, split plot with five replications. The whole plot treatments were four hybrid-planting date combinations consisting of early- or late-planting dates and susceptible or resistant hybrids. The subplot treatments consisted of a factorial arrangement of all possible combinations of three levels of infestation (zero, natural and natural + 3 egg masses) by first brood and the same three levels of infestation by second-brood borers. Temperature and rainfall records were kept at each of the three stations. Borer population and injury to the plant were recorded at the end of the first brood and in the fall. Yield data were collected

Understanding causality and uncertainty in volcanic observations: an example of forecasting eruptive activity on Soufrière Hills Volcano, Montserrat

Following a cessation in eruptive activity it is important to understand how a volcano will behave in the future and when it may next erupt. Such an assessment can be based on the volcano's long-term pattern of behaviour and insights into its current state via monitoring observations. We present a Bayesian network that integrates these two strands of evidence to forecast future eruptive scenarios using expert elicitation. The Bayesian approach provides a framework to quantify the magmatic causes in terms of volcanic effects (i.e., eruption and unrest). In October 2013, an expert elicitation was performed to populate a Bayesian network designed to help forecast future eruptive (in-)activity at Soufrière Hills Volcano. The Bayesian network was devised to assess the state of the shallow magmatic system, as a means to forecast the future eruptive activity in the context of the long-term behaviour at similar dome-building volcanoes. The findings highlight coherence amongst experts when interpreting the current behaviour of the volcano, but reveal considerable ambiguity when relating this to longer patterns of volcanism at dome-building volcanoes, as a class. By asking questions in terms of magmatic causes, the Bayesian approach highlights the importance of using short-term unrest indicators from monitoring data as evidence in long-term forecasts at volcanoes. Furthermore, it highlights potential biases in the judgements of volcanologists and identifies sources of uncertainty in terms of magmatic causes rather than scenario-based outcomes

Effects of Time of Deoxyribonucleic Acid Microinjection on Gene Detection and In Vitro Development of Bovine Embryos

In vivo fertilized embryos were surgically collected from superovulated dairy cows to evaluate microinjection on embryo development and utilized the polymerase chain reaction technique for selection of transgenic embryos. Seventy-two percent of the embryos with visible pronuclei or nuclei were microinjected with DNA, and the remaining 28% served as uninjected controls. All embryos were cocultured with bovine oviductal epithelial cells. Mean final development scores of embryos within the same initial cell stage at collection were unaffected by microinjection. After 144 h of culture, 45% of the microinjected embryos developed to the morula or blastocyst stage. The transgene was detected in 50, 10, and 9% of demimorulae from embryos microinjected at the 1-, 2-, and 4-cell stages. Frequency of transgene detection was higher in morulae from 1-cell embryos than in morulae from 2- and 4-cell embryos. Use of in vitro coculture, embryo bisection, and polymerase chain reaction technique facilitated selection of bovine embryos that carried the transgene

5-HT offsets homeostasis of synaptic transmission during short-term facilitation

Sparks, G. M., and R. L. Cooper. 5-HT offsets homeostasis of synaptic transmission during short-term facilitation. J Appl Physiol 96: 1681-1690, 2004; 10.1152/japplphysiol.01177.2003.-In this study, we approach the topic of vesicle recruitment and recycling by perturbing neurotransmission at the crayfish neuromuscular junction with altered electrical activity and the presence of the neuromodulator serotonin (5-HT). After induction of short-term facilitation (STF) with stimulus pulse trains (40 Hz, 20 pulses), the amount of synaptic transmission can be maintained at a relatively constant level, producing a plateau in the amplitude of the excitatory postsynaptic potentials (EPSPs) throughout the remaining stimuli within a train of a few hundred milliseconds. With an increase in the frequency of the stimuli within a train (60 Hz, 20 pulses), an altered plateau of larger EPSP amplitudes occurs. This suggests that differential rates of vesicle recruitment can be rapidly reached and maintained. Exposure of nerve terminals to 5-HT further enhances the EPSP amplitudes to yet a higher plateau level. The effect of 5-HT is more pronounced for 40-Hz pulse trains than for 60-Hz trains. This suggests that 5-HT can recruit vesicles into the readily releasable pool (RRP) and that the recruitment is limited at higher stimulation frequencies. The attainment of a larger amplitude in the plateaus of the EPSPs at 60 Hz compared with 40 Hz also suggests that the rapid induction of STF enhances the entry of vesicles into the RRP. By direct quantal counts, mean quantal content increases linearly during STF, and 5-HT offsets the linear release. We propose that 5-HT and electrically induced recruitment of vesicles from a reserve pool to the RRP may share similar recruitment mechanisms. synapse; calcium; serotonin; quantal; vesicle REPETITIVE STIMULATION OF presynaptic nerve terminals enhances the amplitude of subsequent excitatory postsynaptic potentials (EPSPs) in a process known as short-term facilitation (STF) The eventual depression of EPSP responses depends on several factors associated with any given synaptic connection, which we do not address here. The persistence of a plateau in EPSP amplitude marks an equilibrium and steady state among synaptic processes. If the frequency of stimulation increases within a pulse train, a new plateau and equilibrium can be observed. Depending on the type of preparation, the electrochemical driving gradient may be sufficient to produce larger EPSP responses during this plateau period. Such is the case for the neuromuscular junction (NMJ) preparations of the crayfish presented in this study. The intracellular calcium concentration ([Ca 2ϩ ] i ) increases within the presynaptic terminal during the stimulation train, and presumably the continual influx, buffering, and exchange mechanisms reach an equilibrium that produces a steady state of transmitter release (9, 16, 25, 28-31, 33, 66, 67). In crustacean NMJs, increasing the frequency of stimulation leads to a new equilibrium with a higher [Ca 2ϩ ] i , and the EPSPs reach the new plateau more quickly than with lower stimulation frequencies (15). As the stimulation frequency is increased, the increase in the maximum plateau response becomes less pronounced. This process is likely regulated by a [Ca 2ϩ ] i -induced enhancement of a Na ϩ /Ca 2ϩ exchanger, which can result in a steady-state process that regulates [Ca 2ϩ ] i effectively during repetitive stimulation. The number of vesicles that can be released at the active zones (AZs) might also be limited. Such an anatomic limitation could explain why higher output terminals associated with phasic motor nerves do not facilitate as much as lower output tonic motor nerve terminals (9), as well as why there are differences in the synaptic efficacy of low-and high-output tonic terminals (16). In this study, we address what effect a faster induction of STF has on the plateau of the EPSP amplitudes to address whether an equilibrium in vesicle recycling is one of the mechanisms that could account for the phenomena. Neuromodulators that offset synaptic function offer an approach to experimentally address the mechanisms underlying differences in facilitation caused by changes in stimulation frequency and inherent differences among synapses. The neuromodulator serotonin (5-HT), which is endogenous to the hemolymph of crustaceans, greatly enhances transmitter release (24) presynaptically at NMJs by increasing the probability of vesicular fusion (54). In crayfish motoneurons, 5-HT mediates its rapid effect through the inositol 1,4,5-trisphosphate (IP 3 ) second messenger system In this report, we show that the equilibrium of vesicle docking and release during the steady plateau phase produced by short trains of stimulation is not limited by the availability of synaptic vesicles, because exposure to 5-HT further enhances vesicular release. METHODS All experiments were performed using the first and second walking legs of freshly obtained crayfish, Procambarus clarkii, measuring 6-10 cm in body length (Atchafalaya Biological Supply, Raceland, LA). Animals were housed individually in an aquatic facility and fe