Tricaine and isoeugenol co-operate towards healthier immobilization.

<p><b>(A)</b> Heat map of percent immobile for 48 combinations of tricaine (0–200 μg/ml) and isoeugenol (0–0.003% v/v). Embryos were dechorionated and soaked from 24–27 hpf when they were assayed for immobility. <b>(B)</b> Continuation of treatments from (A), embryos were assayed for immobility at 72 hpf. <b>(C,D)</b> Representative micrographs of control (C) and 200 μg/ml tricaine treated (D) embryos. Arrow in (D) shows failure of semicircular canal projection fusion. Asterisk in (D) shows pericardial edema. <b>(E)</b> Heat map of percent of embryos with pericardial edema at 72 hpf. <b>(F)</b> Percent control otic vesicle diameter (OVD) was calculated by dividing the average of 10–30 experimental embryos by the average of 10–30 control embryos. Heat map of percent control OVD for the combinatorial treatments. OVD was measured at 72 hpf using micrographs like those in (C, D). Percentage is based on normalization to untreated control. <b>(G)</b> Merge of heatmaps from (A, 27 hpf) and (F) that highlights the tradeoffs between embryo immobility and healthy development.</p

α-bungarotoxin immobilizes embryos while permitting normal development.

<p><b>(A)</b> Percent of embryos immobile after injection of α-bungarotoxin protein (0.046–4.6ng) into the yolk at 24 hpf. <b>(B)</b> Percent of embryos immobile after injection of α-bungarotoxin mRNA (20–400 pg) into the yolk at 24 hpf. <b>(C)</b> Percent of embryos immobile after injection of of α-bungarotoxin mRNA (5–100 pg) into the 1-cell zygote. <b>(D)</b> Percent control OVD at 72 hpf for injection of α-bungarotoxin mRNA into the 1-cell zygote (green), into the yolk (yellow), and reference anesthetic treatments that permitted long-term immobilization (blue). (*) Not significantly different from control, Mann-Whitney-Wilcoxon two tailed P-value 0.87. (†) Significantly different from control, Mann-Whitney-Wilcoxon two tailed P-value 0.0011. <b>(E, G)</b> Control embryo at 72 hpf that was injected with 50 pg of membrane-citrine mRNA into the 1-cell zygote. <b>(F, H)</b> 72 hpf embryo that was injected with 50 pg of α-bungarotoxin mRNA into the 1-cell zygote. <b>(I)</b> Control larva at 8 days post fertilization (dpf) injected with 50 pg of membrane-citrine mRNA into the 1-cell zygote. <b>(J)</b> 8 dpf larva that was injected with 50 pg of α-bungarotoxin mRNA into the 1-cell zygote.</p

Prolonged immobilization with α-bungarotoxin mRNA does not grossly alter neural or cardiovascular development.

<p><b>(A-C)</b> 3D reconstructions of confocal images of 72 hpf <i>Tg(mnx1</i>:<i>gfp)</i> centered at the sixth somite reveal no gross abnormalities in motor neuron patterning or development when embryos are immobilized with 50 pg of α-bungarotoxin mRNA injected at the 1-cell stage <b>(B)</b> or 200 μg/ml of tricaine from 24 to 72 hpf <b>(C)</b> (scale bar 50 μm). A stereotyped axon (red bracket, <b>A</b>, schematic, <b>D</b>) was used to quantify axon branching. <b>(E-G)</b> The distributions of distances between axon branches were not significantly altered in embryos immobilized with 50 pg of α-bungarotoxin mRNA injected at the 1-cell stage <b>(F,</b> Mann-Whitney-Wilcoxon two tailed P-value 0.58<b>)</b> or 200 μg/ml of tricaine from 24 to 72 hpf <b>(G,</b> Mann-Whitney-Wilcoxon two tailed P-value 0.36<b>)</b>. <b>(H)</b> Representative laser-scanning velocimetry results reveal no gross difference in cardiovascular performance between control embryos and embryos immobilized with 50 pg of α-bungarotoxin mRNA. 200 μg/ml of tricaine from 24 to 72 hpf does grossly alter blood flow. Vertical scale bar 100 ms, horizontal scale bar 20 μm. <b>(I-P)</b> Quantitative image analysis of laser-scanning velocimetry reveals no significant difference in cardiovascular function between control and α-bungarotoxin mRNA injected embryos while prolonged tricaine treatment significantly reduces peak blood velocity and peak blood acceleration (Mann-Whitney-Wilcoxon two tailed P-values < 1e-12, tricaine relative to control peak velocities, and <1.4e-11, tricaine relative to control peak accelerations).</p

Algorithm-enabled quantification of cell dynamics during somite formation.

<p>Retrospective cell tracing of epithelial (yellow) and mesenchymal (red) cells from formed somites at (B) 5ss back to the presomitic mesoderm at (A) 3ss. (C) Corresponding decrease in somite tissue surface area during the formation of somites 3, 4, and 5. (D) Epithelial and mesenchymal cell numbers in respective somites at 5ss. (E,F) Three-dimensional cell shape quantified by the length of their principal axes at 3ss and 5ss. (G,H) Scatter plots of elongation () and cell volumes at 3ss and 5ss. The two cell populations show different behavior. Statistical analysis of the two distributions show that mesenchymal cells (red) tend to cluster, round-up, and shrink in size on average.</p

Robust correspondence of automated membrane segmentations with automated nuclear segmentations.

<p>Detection and error rates of the automated algorithm was compared with standard nuclear segmentation algorithms. The assumption was that perfect segmentations of both algorithms should theoretically establish a one-to-one correspondence between nuclei and membranes detected. <b>Matched</b> refers to cells with membrane and nuclei in exact correspondence. <b>Unmatched Cells</b> refer to membranes that did not contain a unique nucleus. <b>Unmatched Nuclei</b> refer to nuclei that did not correspond to a cell membrane.</p

Geometric structure classification based on eigen-system.

<p>An overview of the local intensity structures determined by their eigen-system. Parameters <b>A</b>, <b>B</b>, and <b>S</b> refer to individual terms in the planarity filter (<a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1002780#pcbi.1002780.e055" target="_blank">Equation 1</a> and <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1002780#pcbi.1002780.e056" target="_blank">2</a>). These terms are specified as ratios of individual eigenvalues to enhance the identification of planes relative to rods and ball structure classes.</p

Long-term imaging of embryos immobilized with α-bungarotoxin mRNA.

<p><b>(A)</b> Montage of an immobilized embryo’s development from the 1-cell stage to 85 hpf after it had been injected with 50 pg of α-bungarotoxin mRNA into the 1-cell. Images are shown from every hour of development. <b>(B)</b> Quantification of the full time-course that included 153,452 images that were acquired every 2 seconds. The movement index was calculated as the maximum difference between each image and its subsequent image in the time-series. The index was normalized to the average maximum difference in the first 2,000 time points. Control embryos (red) and embryos in 200 μg/ml tricaine (blue) begin twitching at around 18 hpf and then may swim out of the field while α-bungarotoxin injected embryos (green) showed very little movement until 80 hpf.</p

Accurate and highly-sensitive algorithm performance on synthesized 3D membrane images.

<p>(A–C) Synthesized cell structures in along , and sections with image noise added (<a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1002780#pcbi-1002780-t002" target="_blank">Table 2</a>). As in the case of real-world images, the lateral resolution significantly differs from the axial resolution. (D–F) Segmentations overlaid on the raw image with a 50% opacity function. (G) An example of under-segmentation (brown cells, black arrows) and over-segmentation (interstitial fragments, white arrows) in the image. The errors could be filtered out by size criteria.</p

Scale exploration demonstrates robust algorithm performance.

<p>Precision and recall measures are plotted against different settings of (A) , and (B) , . Precision and recall values were maximized with and and and gradually decreased over broad range of parameter settings indicating robustness. Low scale settings generated noisy features leading to higher over-segmentation rates while large scale settings tended to smooth out sharp membrane corners and cause under-segmentation errors.</p

High-fidelity reconstruction of zebrafish membrane images.

<p>Significant improvement in membrane signal quality is shown in XY, XZ and YZ planes. (A–D) Raw data showing dorsal view (anterior on top) of zebrafish neuroepithelium (ne) and notochord at 12 hpf, (E–H) Planarity function intermediate output and (I–L) Tensor voting final output. The last image in each panel shows a color-mapped zoomed view for easy comparison.</p