Through the injection of f-aequorin (a calcium-specific luminescent reporter) and the use of an imaging photon detector, we see a distinct localized elevation of intracellular calcium that accompanies the appearance of the first furrow arc at the blastodisc surface: the furrow positioning signal. As the leading edges of the arc progress outward toward the margins of the blastodisc, they are accompanied by two subsurface slow calcium waves moving at about 0.2 micron/s: the furrow propagation signal. As these wave fronts approach the edge of the blastodisc, another calcium signal arises in the central region where the positioning signal originally appeared. Like the propagation signal, it extends outward to the margins of the blastodisc, but in this case it also moves downward, accompanying the deepening process that separates the daughter cells: the furrow deepening signal. Both of these furrow deepening progressions move at around 0.1 to 0.2 micron/s. The deepening signal begins to diminish from the center outward, returning to precleavage resting levels on completion of cytokinesis. The signaling sequence is repeated during the second cell division cycle. These localized transients do not require external calcium and they can be dissipated after they have begun by introducing calcium shuttle buffers, resulting in furrow delocalization and regression. They also occur in parthenogenetically activated eggs in which, in an attenuated form, they accompany abortive cleavages.

Oscillations of cytosolic free calcium levels have been shown to inf luence gene regulation and cell differentiation in a variety of model systems. Intercellular calcium waves thus present a plausible mechanism for coordinating cellular processes during embryogenesis. Herein we report use of aequorin and a photon imaging microscope to directly observe a rhythmic series of intercellular calcium waves that circumnavigate zebrafish embryos over a 10-h period during gastrulation and axial segmentation. These waves first appeared at about 65% epiboly and continued to arise every 5-10 min up to at least the 16-somite stage. The waves originated from loci of high calcium activity bordering the blastoderm margin. Several initiating loci were active early in the wave series, whereas later a dorsal marginal midline locus predominated. On completion of epiboly, the dorsal locus was incorporated into the developing tail bud and continued to generate calcium waves. The locations and timing at which calcium dynamics are most active appear to correspond closely to embryonic cellular and syncytial sites of known morphogenetic importance. The observations suggest that a panembryonic calcium signaling system operating in a clock-like fashion might play a role during vertebrate axial patterning.The vertebrate body plan emerges during gastrulation through patterns of inductive interactions, cellular rearrangements, and gene expression, some of which may be coordinated across large distances with considerable temporal precision (1-3). Morphogen gradients and propagating second messenger waves, especially those involving calcium and inositol 1,4,5-trisphosphate, have been proposed as carriers of such putative long-range coordinating messages (4-7). Intercellular calcium waves are of particular interest because oscillations of intracellular calcium levels have been shown to directly influence expression of numerous developmentally regulated genes in cultured cells (8,9). To search for evidence of possible large-scale embryonic signaling mechanisms, we imaged whole-embryo calcium dynamics in an optically suitable vertebrate, the zebrafish (Danio rerio), by using a bioluminescent calcium reporter. An imaging system using a photon-counting spatial detector allowed for long-term imaging of calciumtriggered luminescence with high temporal and moderate spatial resolution (16-kHz sampling rate; spatial coordinate units ϭ 12 m 2 with a ϫ10 objective). Because at this spatial scale, light arising from cells well above and below the nominal object plane still gave useful imaging information, an effective imaging field depth of 100-200 m was achieved. A surprising array of spatially and temporally complex calcium transients was imaged with this technique. Herein we report initial observations on the most unusual of these patterns, a rhythmic series of intercellular calcium waves that traverse the blastoderm margin and main body axis during gastrulation and axial segmentation.Zebrafish eggs were injected 10 min after fertilization with 10...

In this paper, we report on the investigation of silicon avalanche photodiodes (APDs) for high-energy photon imaging applications. This includes a new APD design that provides X-ray and-ray imaging with significant reduction in electronic readout requirements. This new APD design, referred to as position-sensitive avalanche photodiode (PSAPD), involves charge sharing amongst the electrodes that enable determination of position of interaction. PSAPDs with 14 14 mm 2 area have been fabricated using planar processing. The performance of these devices has been evaluated for energy resolution, timing resolution (4 ns full-width at half-maximum), and spatial resolution (300 m intrinsic spatial resolution). The potential of these APDs in high-energy physics and medical imaging is addressed.

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The construction and application of genetically encoded intracellular calcium concentration ([Ca2+]i) indicators has a checkered history. Excitement raised over the creation of new probes is often followed by disappointment when it is found that the initial demonstrations of [Ca2+]i sensing capability cannot be leveraged into real scientific advances. Recombinant apo-aequorin cloned from Aequorea victoria was the first Ca2+ sensitive protein genetically targeted to subcellular compartments. In the jellyfish, bioluminescence resonance energy transfer (BRET) between Ca2+ bound aequorin and green fluorescent protein (GFP) emits green light. Similarly, Ca2+ sensitive bioluminescent reporters undergoing BRET have been constructed between aequorin and GFP, and more recently with other fluorescent protein variants. These hybrid proteins display red-shifted spectrums and have higher light intensities and stability compared to aequorin alone. We report BRET measurement of single-cell [Ca2+]i based on the use of electron-multiplying charge-coupled-detector (EMCCD) imaging camera technology, mounted on either a bioluminescence or conventional microscope. Our results show for the first time how these new technologies make facile long-term monitoring of [Ca2+]i at the single-cell level, obviating the need for expensive, fragile, and sophisticated equipment based on image-photon-detectors (IPD) that were until now the only technical recourse to dynamic BRET experiments of this type.