Design and test of Multibow in zebrafish.

<p><b>a.</b> Modified “Brainbow [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0127822#pone.0127822.ref001" target="_blank">1</a>]” cassette that allows a binary ON/OFF switch. <b>b.</b> Multibow Strategy. Each cell harbors multiple different ON/OFF cassettes to generate random color “digital” barcodes upon Cre-mediated recombination. <b>c.</b> Table of Multibow Tags and Fluorescent Proteins (FPs). <b>d.</b> Diversity of color codes. Image is a densely labeled region along the trunk of a 40hpf <i>hsp70</i>:<i>cerulean-cre</i> embryo injected with all 21 Multibow constructs and heat-shocked at 10hpf for 1 hour. The color and tag diversity generates barcodes for cell clones that appear random and diverse. Intensity differences further help distinguish cells from neighbors visually. The Composite image is made from the green, yellow (turned to blue) and red panels. 3 different clones are highlighted by α, β, γ and corresponding arrows. Scale bar: 10μm. See also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0127822#pone.0127822.s006" target="_blank">S3 Table</a>. <b>e.</b> Partial table of clones of different color codes found in <b>d.</b>. The colored square labels of the top row indicate nuclear, membrane and cytoplasmic, respectively. A black square in the table indicates this clone being positive for the corresponding color. Distinct "barcodes" form for different clones. The α, β, γ clones are indicated by arrows. The number of annotated cells labels (~30) represents a large fraction of cells found in the image in <b>d</b>, which contains ~50 cells. The fact that most of these cells have a color code distinct from any other cell (except clones that have the same color) show that Multibow label is highly random.</p

Spatial temporal coverage and stability of Multibow labeling.

<p><b>a.</b> Spatial and cell type coverage of Multibow. The embryo was injected with 6 Multibow colors (mR/mG/nR/nG/R/G) at single cell stage and heat-shocked at 1 day-post-fertilization (dpf) for 2 hours. The whole 4dpf larva was imaged in 2 channels (G/R). Positive cells can be seen distributed from head to tail throughout the larva, indicating high spatial coverage. In inserts 1 and 2, distinctly shaped skin, muscle, mesenchymal and neural cells can be observed by cytoplasmic or membrane Multibow labeling. Scale bars: 100μm. <b>b.</b> Temporal stability of labeling. The embryo was injected with 6 Multibow colors (mR/mG/nR/nG/R/G) at single cell stage and heat-shocked at 1 day-post-fertilization (dpf) for 2 hours. The same embryo was imaged once per day to 11dpf. The persistence of labeling indicates genomic insertion of Multibow cassettes. Red patches around the eye and along the gut are auto-fluorescence. Enlarged views of white boxed areas show that the area is stably fluorescent. Scale bar in enlarged views: 100μm. <b>c.</b> Label stability of color codes over time. The embryo was injected with 12 (B/G/Y/R) Multibow constructs at one cell stage. Heat-shock of this tg(<i>hsp70</i>:<i>cerulean-cre</i>) individual was at 30hpf (duration: 2 hours). Its developing larval tail fin was imaged every 24 hours starting at 54hpf using four channels (B/G/Y/R). The color codes of the cells remain unchanged despite fluorescent intensity differences at different days, allowing identification of the same cells/clones(e.g., α and β, shown in enlarged regions marked by white boxes). Color codes: α: nG/nY; β: mB. Scale bar: 100μm. See also Fig d in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0127822#pone.0127822.s002" target="_blank">S2 Fig</a>.</p

Examples of Multibow Cell Tracing in Development and Regeneration.

<p><b>a.</b> Cranial facial development mapped by Multibow. The embryo was heat-shocked at 6hpf. 4 channels (B/G/Y/R) were used. The left face of the larva was imaged. Red boxes: regions highlighted in <b>b.</b> and <b>c.</b>. Scale bars: 50μm. <b>b.</b> Lineage relationship between neuromast hair cells. Dashed line circle indicates the hair bundle. Multibow labeled hair cell color codes: 1(mB/nY/R), 2(mB/mG/nR), 3(mB), 4(nG), 5(mB/nR), 6(R). The same pattern was already observed at 30hpf. Scale bars: 10μm. <b>c.</b> Identification of cells that undergo remarkable morphological changes during semicircular canal formation. Arrows: initial locations of the two mesenchymal cells that span the projection later. Grey circle: posterior otolith. Scale bars: 50μm. <b>d.</b> Clonal expansion near the eye over long time periods. The embryo was injected with 12 constructs (B/G/Y/R) and heat-shocked at 10hpf. Arrows indicate locations of identified clones α (nG), β (nG/R), γ (nY/mR). These clones can be seen amplified in number at 54hpf or 129hpf (α: 2 to 4; β: 2 to 4; γ: 2 to 3). Scale bar: 100μm. <b>e.</b> Multibow analysis of regeneration in the larval tail. Heat-shock labeling (1 hour), amputation and imaging were performed as labeled in the timeline. Immediately after amputation, the tissue shrank and cells near the wound converged (the images overlay may appear to be slightly out of register due to the changes of the live tissue during the acquisition of different channels, cell identification is not affected as these changes are small and predictable). By 2 days after amputation, most cells that had converged at the frontier of the wound were gone (their unique color codes disappeared, red arrowheads). The regenerated tissue came from clonal expansion of cells away from the frontier (highlighted examples in enlarged view from the white boxes 1 and 2). These clones show lineage restriction to the original cell type (the morphology of cells in the same clone remains similar, e.g., the blue cells in box 1 increased in number while size and shape do not have major changes.). Scale bars: 50μm.</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

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

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

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

Reconstructing the membrane signal by eliminating intensity inhomogeneities.

<p>A single cell membrane is shown across (A) , (B) , and (C) sections. The plane shows a consistently bold and uniform membrane signal while the and views show a non-uniform membrane signal. Membrane planes <i>en-face</i> to the optical planes (marked by red arrows) are very weak and markedly diffuse in intensity. Membranes orthogonal to the imaging plane are sharper (blue arrows). (D) A qualitative model describing the formation of a membrane image under a fluorescent microscope. Flourophores tagged to membranes are shown as a point cloud (input). The focal planes are shown in red and the obtained intensity profiles on the plane are shown as plots. Cell membranes imaged oblique and <i>en face</i> such as the interface between cells are poorly visible in comparison to those orthogonal to the focal planes. (E) Three stages in the reconstruction process: (i) Detect membrane planes by mining for planar fluorophore distributions. This allows even weak membranes (<i>en-face</i> or oblique) to be extracted and accounts for intensity inhomogeneity. (ii) Voting to fill structural gaps or holes in the membrane signal that may not be contiguous. (iii) Region segmentation using the watershed algorithm to extract three-dimensional cell meshes for quantification.</p