Local delivery of fluorescent dye for fiber-optics confocal microscopy of the living heart

Fiber-optics confocal microscopy (FCM) is an emerging imaging technology with various applications in basic research and clinical diagnosis. FCM allows for real-time in situ microscopy of tissue at sub-cellular scale. Recently FCM has been investigated for cardiac imaging, in particular, for discrimination of cardiac tissue during pediatric open-heart surgery. FCM relies on fluorescent dyes. The current clinical approach of dye delivery is based on systemic injection, which is associated with high dye consumption, and adverse clinical events. In this study, we investigated approaches for local dye delivery during FCM imaging based on dye carriers attached to the imaging probe. Using three-dimensional confocal microscopy, automated bench tests, and FCM imaging we quantitatively characterized dye release of carriers composed of open-pore foam only and foam loaded with agarose hydrogel. In addition, we compared local dye delivery with a model of systemic dye delivery in the isolated perfused rodent heart. We measured the signal-to-noise ratio (SNR) of images acquired in various regions of the heart. Our evaluations showed that foam-agarose dye carriers exhibited a prolonged dye release vs. foam-only carriers. Foam-agarose dye carriers allowed reliable imaging of 5–9 lines, which is comparable to 4–8 min of continuous dye release. Our study in the living heart revealed that the SNR of FCM images using local and systemic dye delivery is not different. However, we observed differences in the imaged tissue microstructure with the two approaches. Structural features characteristic of microvasculature were solely observed for systemic dye delivery. Our findings suggest that local dye delivery approach for FCM imaging constitutes an important alternative to systemic dye delivery. We suggest that the approach for local dye delivery will facilitate clinical translation of FCM, for instance, for FCM imaging during pediatric heart surgery

Modeling effects of L-type ca(2+) current and na(+)-ca(2+) exchanger on ca(2+) trigger flux in rabbit myocytes with realistic T-tubule geometries.

The transverse tubular system of rabbit ventricular myocytes consists of cell membrane invaginations (t-tubules) that are essential for efficient cardiac excitation-contraction coupling. In this study, we investigate how t-tubule micro-anatomy, L-type Ca(2+) channel (LCC) clustering, and allosteric activation of Na(+)/Ca(2+) exchanger by L-type Ca(2+) current affects intracellular Ca(2+) dynamics. Our model includes a realistic 3D geometry of a single t-tubule and its surrounding half-sarcomeres for rabbit ventricular myocytes. The effects of spatially distributed membrane ion-transporters (LCC, Na(+)/Ca(2+) exchanger, sarcolemmal Ca(2+) pump, and sarcolemmal Ca(2+) leak), and stationary and mobile Ca(2+) buffers (troponin C, ATP, calmodulin, and Fluo-3) are also considered. We used a coupled reaction-diffusion system to describe the spatio-temporal concentration profiles of free and buffered intracellular Ca(2+). We obtained parameters from voltage-clamp protocols of L-type Ca(2+) current and line-scan recordings of Ca(2+) concentration profiles in rabbit cells, in which the sarcoplasmic reticulum is disabled. Our model results agree with experimental measurements of global Ca(2+) transient in myocytes loaded with 50 μM Fluo-3. We found that local Ca(2+) concentrations within the cytosol and sub-sarcolemma, as well as the local trigger fluxes of Ca(2+) crossing the cell membrane, are sensitive to details of t-tubule micro-structure and membrane Ca(2+) flux distribution. The model additionally predicts that local Ca(2+) trigger fluxes are at least threefold to eightfold higher than the whole-cell Ca(2+) trigger flux. We found also that the activation of allosteric Ca(2+)-binding sites on the Na(+)/Ca(2+) exchanger could provide a mechanism for regulating global and local Ca(2+) trigger fluxes in vivo. Our studies indicate that improved structural and functional models could improve our understanding of the contributions of L-type and Na(+)/Ca(2+) exchanger fluxes to intracellular Ca(2+) dynamics

The banana code—natural blend processing in the olfactory circuitry of Drosophila melanogaster

Odor information is predominantly perceived as complex odor blends. For Drosophila melanogaster one of the most attractive blends is emitted by an over-ripe banana. To analyze how the fly's olfactory system processes natural blends we combined the experimental advantages of gas chromatography and functional imaging (GC-I). In this way, natural banana compounds were presented successively to the fly antenna in close to natural occurring concentrations. This technique allowed us to identify the active odor components, use these compounds as stimuli and measure odor-induced Ca(2+) signals in input and output neurons of the Drosophila antennal lobe (AL), the first olfactory neuropil. We demonstrate that mixture interactions of a natural blend are very rare and occur only at the AL output level resulting in a surprisingly linear blend representation. However, the information regarding single components is strongly modulated by the olfactory circuitry within the AL leading to a higher similarity between the representation of individual components and the banana blend. This observed modulation might tune the olfactory system in a way to distinctively categorize odor components and improve the detection of suitable food sources. Functional GC-I thus enables analysis of virtually any unknown natural odorant blend and its components in their relative occurring concentrations and allows characterization of neuronal responses of complete neural assemblies. This technique can be seen as a valuable complementary method to classical GC/electrophysiology techniques, and will be a highly useful tool in future investigations of insect-insect and insect-plant chemical interactions

Third-order neurons in the lateral horn enhance bilateral contrast of odor inputs through contralateral inhibition in Drosophila

The survival and reproduction of Drosophila melanogaster depends heavily on its ability to determine the location of an odor source and either to move towards or away from it. Despite the very small spatial separation between the two antennae and the redundancy in sensory neuron projection to both sides of the brain, Drosophila can resolve the concentration gradient by comparing the signal strength between the two antennae. When an odor stimulates the antennae asymmetrically, ipsilateral projection neurons from the first olfactory center are more strongly excited compared to the contralateral ones. However, it remains elusive how higher-order neurons process such asymmetric or lateralized odor inputs. Here, we monitored and analyzed for the first time the activity patterns of a small cluster of third-order neurons (so-called VLP neurons) to asymmetric olfactory stimulation using two-photon calcium imaging. Our data demonstrate that lateralized odors evoke distinct activation of these neurons in the left and right brain hemisphere as a result of contralateral inhibition. Moreover, using laser transection experiments we show that this contralateral inhibition is mediated by presynaptic neurons most likely located in the lateral horn. Finally, we propose that this inhibitory interaction between higher-order neurons facilitates odor lateralization and plays a crucial role in olfactory navigation behavior of Drosophila, a theory that needs to be experimentally addressed in future studies

Impact of multiscale dynamical processes and mixing on the chemical composition of the upper troposphere and lower stratosphere during the Intercontinental Chemical Transport Experiment–North America

We use high-frequency in situ observations made from the DC8 to examine fine-scale tracer structure and correlations observed in the upper troposphere and lower stratosphere during INTEX-NA. Two flights of the NASA DC-8 are compared and contrasted. Chemical data from the DC-8 flight on 18 July show evidence for interleaving and mixing of polluted and stratospheric air masses in the vicinity of the subtropical jet in the upper troposphere, while on 2 August the DC-8 flew through a polluted upper troposphere and a lowermost stratosphere that showed evidence of an intrusion of polluted air. We compare data from both flights with RAQMS 3-D global meteorological and chemical model fields to establish dynamical context and to diagnose processes regulating the degree of mixing on each day. We also use trajectory mapping of the model fields to show that filamentary structure due to upstream strain deformation contributes to tracer variability observed in the upper troposphere. An Eulerian measure of strain versus rotation in the large-scale flow is found useful in predicting filamentary structure in the vicinity of the jet. Higher-frequency (6–24 km) tracer variability is attributed to buoyancy wave oscillations in the vicinity of the jet, whose turbulent dissipation leads to efficient mixing across tracer gradients

Trace chemical measurements from the northern midlatitude lowermost stratosphere in early spring: Distributions, correlations, and fate

In situ measurements of a large number of trace chemicals from the midlatitude (37-57°N) lower stratosphere were performed with the NASA DC-8 aircraft during March 1994. Deepest penetrations into the stratosphere (550 ppb O3, 279 ppb N2O, and 350 K potential temperature) corresponded to a region that has been defined as the "lowermost stratosphere" (LS) by Holton et al [1995]. Analysis of data shows that the mixing ratios of long-lived tracer species (e. g. CH4, HNO3, NOy, CFCs) are linearly correlated with those of O3 and N2O. A ΔNOy/ΔO3 of 0.0054 ppb/ppb and ΔNOy/ΔN2O of -0.081 ppb/ppb is in good agreement with other reported measurements from the DC-8. These slopes are however, somewhat steeper than those reported from the ER-2 airborne studies. We find that the reactive nitrogen budget in the LS is largely balanced with HNO3 accounting for 80% of NOy, and PAN and NOx together accounting for 5%. A number of oxygenated species (e. g. acetone, H2O2) were present and may provide an important in situ source of HOx in the LS. SO2 mixing ratios were found to increase in the stratosphere at a rate that was comparable to the decline in OCS levels. No evidence of particle formation could be observed. Ethane, propane, and acetylene mixing ratios declined rapidly in the LS with Cl atoms likely playing a key role in this process. A number of reactive hydrocarbons/halocarbons (e. g. C6H6, CH3I) were present at low but measurable concentrations