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A hybrid blob-slice model for accurate and efficient detection of fluorescence labeled nuclei in 3D.

Santella A, Du Z, Nowotschin S, Hadjantonakis AK, Bao Z - BMC Bioinformatics (2010)

Bottom Line: Because our approach is specialized for the characteristics of optically sectioned nuclear images, it can achieve superior accuracy in significantly less time than other approaches.Our approach is fast, accurate, available as open source software and its learned shape model is easy to retrain.As our pharynx development example shows, these characteristics make single cell analysis relatively easy and will enable novel experimental methods utilizing complex data sets.

View Article: PubMed Central - HTML - PubMed

Affiliation: Developmental Biology, Sloan-Kettering Institute, 1275 York Avenue, New York, New York 10065, USA.

ABSTRACT

Background: To exploit the flood of data from advances in high throughput imaging of optically sectioned nuclei, image analysis methods need to correctly detect thousands of nuclei, ideally in real time. Variability in nuclear appearance and undersampled volumetric data make this a challenge.

Results: We present a novel 3D nuclear identification method, which subdivides the problem, first segmenting nuclear slices within each 2D image plane, then using a shape model to assemble these slices into 3D nuclei. This hybrid 2D/3D approach allows accurate accounting for nuclear shape but exploits the clear 2D nuclear boundaries that are present in sectional slices to avoid the computational burden of fitting a complex shape model to volume data. When tested over C. elegans, Drosophila, zebrafish and mouse data, our method yielded 0 to 3.7% error, up to six times more accurate as well as being 30 times faster than published performances. We demonstrate our method's potential by reconstructing the morphogenesis of the C. elegans pharynx. This is an important and much studied developmental process that could not previously be followed at this single cell level of detail.

Conclusions: Because our approach is specialized for the characteristics of optically sectioned nuclear images, it can achieve superior accuracy in significantly less time than other approaches. Both of these characteristics are necessary for practical analysis of overwhelmingly large data sets where processing must be scalable to hundreds of thousands of cells and where the time cost of manual error correction makes it impossible to use data with high error rates. Our approach is fast, accurate, available as open source software and its learned shape model is easy to retrain. As our pharynx development example shows, these characteristics make single cell analysis relatively easy and will enable novel experimental methods utilizing complex data sets.

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Reconstruction of C. elegans pharynx development. I. Assembly of the primordium. MSaa and MSpa lineages are in cyan. On the right side ABaraaap (and then its anterior daughter) is in red, ABarapaa in pink, ABarapap in yellow and ABaraapp in blue. On the left side, the symmetric sublineages are shown in the same colors but are marked by arrows, with ABalpaap (and then its anterior daughter) in red, ABalpaaa in pink, ABalpapp in yellow and ABaraapa in blue. White in frame a represents ABaraaaa, which gives rise to two L/R symmetric sublineages (in magenta in frame b and c) as well as a pair of cells one of which undergoes apoptosis and the other of which forms the third fold of symmetry for part of that sublineage (white in frame b and c). Grey represents a non-pharyngeal precursor, ABalpapa which interrupts the left side group at birth (frame a) but is excluded during subsequent development. For all frames in this figure, the non highlighted cells are shown as semi-transparent spheres. In frame a, at time 160, left-right symmetric precursor cells have been born but are not symmetric in their layout. Note the midline marked by the two rows of MS/cyan cells. MS cells have just started to enter the inside of the embryo. The blue cell that is part of the left side is born on the right side of the midline but will cross over to join the other left side cells. In frame b, time 207, the AB pharynx cells have moved to the midline to cover the MS cells. The blue cell of the left group has crossed the midline to assume a symmetrical position as its right counterpart. However, the pink cells of the left group are still disconnected from the yellow cells compared to the right side. The grey non-pharyngeal cells are now excluded from the primordium. In frame c, time 250, the left and right AB groups are fully assembled and symmetrical. II. The inflation of the primordium. To illustrate the topological mapping of the primordium to the mature pharynx, cells are colored as follows: white for buccal cavity, red for the corpus/anterior lobe, blue for the posterior lobe and purple for precursors whose descendents contribute to both lobes. The E/gut cells are shown in green for context. Frame a shows the primordium prior to inflation, where cells are arranged in two flat sheets that are left-right symmetric. In Frame b the sheets have begun to round slightly. In c they have rearranged to create a rounded shape, and the ventral MS portion of the pharynx moved anterior to the E cells. III. The emergence of threefold symmetry. Pharyngeal right side terminal cells (and their precursors) are in blue, those on the left are in red. Terminal cells and precursors are white if they, or their descendents, have no L/R counterpart. These cells make up the third component of the final threefold lumen symmetry. IV. Frame a shows the correspondence between pharynx cells whose lineages are annotated as left right symmetric with a line. A left view, angled slightly posterior-dorsal y, highlights the consistent alignment. Frame b, the position of cells at ~340 min pfc. Frames a and b use the same color scheme as in I with the addition of the E/gut cells in green. Frame c shows the final configuration of the pharynx colored as in II.
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Figure 5: Reconstruction of C. elegans pharynx development. I. Assembly of the primordium. MSaa and MSpa lineages are in cyan. On the right side ABaraaap (and then its anterior daughter) is in red, ABarapaa in pink, ABarapap in yellow and ABaraapp in blue. On the left side, the symmetric sublineages are shown in the same colors but are marked by arrows, with ABalpaap (and then its anterior daughter) in red, ABalpaaa in pink, ABalpapp in yellow and ABaraapa in blue. White in frame a represents ABaraaaa, which gives rise to two L/R symmetric sublineages (in magenta in frame b and c) as well as a pair of cells one of which undergoes apoptosis and the other of which forms the third fold of symmetry for part of that sublineage (white in frame b and c). Grey represents a non-pharyngeal precursor, ABalpapa which interrupts the left side group at birth (frame a) but is excluded during subsequent development. For all frames in this figure, the non highlighted cells are shown as semi-transparent spheres. In frame a, at time 160, left-right symmetric precursor cells have been born but are not symmetric in their layout. Note the midline marked by the two rows of MS/cyan cells. MS cells have just started to enter the inside of the embryo. The blue cell that is part of the left side is born on the right side of the midline but will cross over to join the other left side cells. In frame b, time 207, the AB pharynx cells have moved to the midline to cover the MS cells. The blue cell of the left group has crossed the midline to assume a symmetrical position as its right counterpart. However, the pink cells of the left group are still disconnected from the yellow cells compared to the right side. The grey non-pharyngeal cells are now excluded from the primordium. In frame c, time 250, the left and right AB groups are fully assembled and symmetrical. II. The inflation of the primordium. To illustrate the topological mapping of the primordium to the mature pharynx, cells are colored as follows: white for buccal cavity, red for the corpus/anterior lobe, blue for the posterior lobe and purple for precursors whose descendents contribute to both lobes. The E/gut cells are shown in green for context. Frame a shows the primordium prior to inflation, where cells are arranged in two flat sheets that are left-right symmetric. In Frame b the sheets have begun to round slightly. In c they have rearranged to create a rounded shape, and the ventral MS portion of the pharynx moved anterior to the E cells. III. The emergence of threefold symmetry. Pharyngeal right side terminal cells (and their precursors) are in blue, those on the left are in red. Terminal cells and precursors are white if they, or their descendents, have no L/R counterpart. These cells make up the third component of the final threefold lumen symmetry. IV. Frame a shows the correspondence between pharynx cells whose lineages are annotated as left right symmetric with a line. A left view, angled slightly posterior-dorsal y, highlights the consistent alignment. Frame b, the position of cells at ~340 min pfc. Frames a and b use the same color scheme as in I with the addition of the E/gut cells in green. Frame c shows the final configuration of the pharynx colored as in II.

Mentions: We have reconstructed pharyngeal development up to the stage where structures corresponding to the parts of the fully formed pharynx can be visually identified in the embryo (~340 minutes post first cell cleavage, pfc). Visualized in 3D, early morphogenesis of the pharynx appears to involve two distinct stages. During the first stage, pharyngeal precursor cells are recruited from discrete regions of the embryo to form a coherent structure with an overall left-right symmetry (Figure 5I, Additional file 10: Movie 3). Pharyngeal cells are derived from the AB and MS lineages, with the MS cells born in a contiguous structure and the AB cells assembled piecemeal. In the MS lineage, pharyngeal precursors are born in two rows, one on the left side and one on the right (cyan in Figure 5I). The two rows are born next to each other and the midline corresponds to the future midline of the organ, around which the AB cells assemble. The AB cells can be further divided into two groups. The right side group is derived from the ABara sublineage. Cells in this group (red, pink, yellow and blue in Figure 5I) are born next to each other (Figure 5I frame a) and maintain their relative positions as they move towards the midline to meet the left side group (Figure 5I frames b and c). In contrast, the corresponding cells in the left side group (sharing colors with their left side fate counterparts in Figure 5I but marked with arrows) are born isolated and migrate towards each other to assemble a mirror image of the right side group (Figure 5I frames b and c). In the meantime, the pharyngeal precursors move from the ventral surface to the inside of the embryo. This process starts with the MS cells at around 160 minutes pfc (Figure 5I frame a). The AB cells first move on the ventral surface towards the midline (see above) to cover the MS cells (Figure 5I frame b) before following them inside (Figure 5I frame c). This stage of morphogenesis, which we term the assembly stage, ends at ~250 minutes pfc. The end result is a contiguous primordium consisting of two flat sheets of cells and an overall left-right symmetry. This is highlighted in Figure 5IV frame a, which marks the correspondences between pharyngeal cells from symmetric lineages.


A hybrid blob-slice model for accurate and efficient detection of fluorescence labeled nuclei in 3D.

Santella A, Du Z, Nowotschin S, Hadjantonakis AK, Bao Z - BMC Bioinformatics (2010)

Reconstruction of C. elegans pharynx development. I. Assembly of the primordium. MSaa and MSpa lineages are in cyan. On the right side ABaraaap (and then its anterior daughter) is in red, ABarapaa in pink, ABarapap in yellow and ABaraapp in blue. On the left side, the symmetric sublineages are shown in the same colors but are marked by arrows, with ABalpaap (and then its anterior daughter) in red, ABalpaaa in pink, ABalpapp in yellow and ABaraapa in blue. White in frame a represents ABaraaaa, which gives rise to two L/R symmetric sublineages (in magenta in frame b and c) as well as a pair of cells one of which undergoes apoptosis and the other of which forms the third fold of symmetry for part of that sublineage (white in frame b and c). Grey represents a non-pharyngeal precursor, ABalpapa which interrupts the left side group at birth (frame a) but is excluded during subsequent development. For all frames in this figure, the non highlighted cells are shown as semi-transparent spheres. In frame a, at time 160, left-right symmetric precursor cells have been born but are not symmetric in their layout. Note the midline marked by the two rows of MS/cyan cells. MS cells have just started to enter the inside of the embryo. The blue cell that is part of the left side is born on the right side of the midline but will cross over to join the other left side cells. In frame b, time 207, the AB pharynx cells have moved to the midline to cover the MS cells. The blue cell of the left group has crossed the midline to assume a symmetrical position as its right counterpart. However, the pink cells of the left group are still disconnected from the yellow cells compared to the right side. The grey non-pharyngeal cells are now excluded from the primordium. In frame c, time 250, the left and right AB groups are fully assembled and symmetrical. II. The inflation of the primordium. To illustrate the topological mapping of the primordium to the mature pharynx, cells are colored as follows: white for buccal cavity, red for the corpus/anterior lobe, blue for the posterior lobe and purple for precursors whose descendents contribute to both lobes. The E/gut cells are shown in green for context. Frame a shows the primordium prior to inflation, where cells are arranged in two flat sheets that are left-right symmetric. In Frame b the sheets have begun to round slightly. In c they have rearranged to create a rounded shape, and the ventral MS portion of the pharynx moved anterior to the E cells. III. The emergence of threefold symmetry. Pharyngeal right side terminal cells (and their precursors) are in blue, those on the left are in red. Terminal cells and precursors are white if they, or their descendents, have no L/R counterpart. These cells make up the third component of the final threefold lumen symmetry. IV. Frame a shows the correspondence between pharynx cells whose lineages are annotated as left right symmetric with a line. A left view, angled slightly posterior-dorsal y, highlights the consistent alignment. Frame b, the position of cells at ~340 min pfc. Frames a and b use the same color scheme as in I with the addition of the E/gut cells in green. Frame c shows the final configuration of the pharynx colored as in II.
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Related In: Results  -  Collection

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Figure 5: Reconstruction of C. elegans pharynx development. I. Assembly of the primordium. MSaa and MSpa lineages are in cyan. On the right side ABaraaap (and then its anterior daughter) is in red, ABarapaa in pink, ABarapap in yellow and ABaraapp in blue. On the left side, the symmetric sublineages are shown in the same colors but are marked by arrows, with ABalpaap (and then its anterior daughter) in red, ABalpaaa in pink, ABalpapp in yellow and ABaraapa in blue. White in frame a represents ABaraaaa, which gives rise to two L/R symmetric sublineages (in magenta in frame b and c) as well as a pair of cells one of which undergoes apoptosis and the other of which forms the third fold of symmetry for part of that sublineage (white in frame b and c). Grey represents a non-pharyngeal precursor, ABalpapa which interrupts the left side group at birth (frame a) but is excluded during subsequent development. For all frames in this figure, the non highlighted cells are shown as semi-transparent spheres. In frame a, at time 160, left-right symmetric precursor cells have been born but are not symmetric in their layout. Note the midline marked by the two rows of MS/cyan cells. MS cells have just started to enter the inside of the embryo. The blue cell that is part of the left side is born on the right side of the midline but will cross over to join the other left side cells. In frame b, time 207, the AB pharynx cells have moved to the midline to cover the MS cells. The blue cell of the left group has crossed the midline to assume a symmetrical position as its right counterpart. However, the pink cells of the left group are still disconnected from the yellow cells compared to the right side. The grey non-pharyngeal cells are now excluded from the primordium. In frame c, time 250, the left and right AB groups are fully assembled and symmetrical. II. The inflation of the primordium. To illustrate the topological mapping of the primordium to the mature pharynx, cells are colored as follows: white for buccal cavity, red for the corpus/anterior lobe, blue for the posterior lobe and purple for precursors whose descendents contribute to both lobes. The E/gut cells are shown in green for context. Frame a shows the primordium prior to inflation, where cells are arranged in two flat sheets that are left-right symmetric. In Frame b the sheets have begun to round slightly. In c they have rearranged to create a rounded shape, and the ventral MS portion of the pharynx moved anterior to the E cells. III. The emergence of threefold symmetry. Pharyngeal right side terminal cells (and their precursors) are in blue, those on the left are in red. Terminal cells and precursors are white if they, or their descendents, have no L/R counterpart. These cells make up the third component of the final threefold lumen symmetry. IV. Frame a shows the correspondence between pharynx cells whose lineages are annotated as left right symmetric with a line. A left view, angled slightly posterior-dorsal y, highlights the consistent alignment. Frame b, the position of cells at ~340 min pfc. Frames a and b use the same color scheme as in I with the addition of the E/gut cells in green. Frame c shows the final configuration of the pharynx colored as in II.
Mentions: We have reconstructed pharyngeal development up to the stage where structures corresponding to the parts of the fully formed pharynx can be visually identified in the embryo (~340 minutes post first cell cleavage, pfc). Visualized in 3D, early morphogenesis of the pharynx appears to involve two distinct stages. During the first stage, pharyngeal precursor cells are recruited from discrete regions of the embryo to form a coherent structure with an overall left-right symmetry (Figure 5I, Additional file 10: Movie 3). Pharyngeal cells are derived from the AB and MS lineages, with the MS cells born in a contiguous structure and the AB cells assembled piecemeal. In the MS lineage, pharyngeal precursors are born in two rows, one on the left side and one on the right (cyan in Figure 5I). The two rows are born next to each other and the midline corresponds to the future midline of the organ, around which the AB cells assemble. The AB cells can be further divided into two groups. The right side group is derived from the ABara sublineage. Cells in this group (red, pink, yellow and blue in Figure 5I) are born next to each other (Figure 5I frame a) and maintain their relative positions as they move towards the midline to meet the left side group (Figure 5I frames b and c). In contrast, the corresponding cells in the left side group (sharing colors with their left side fate counterparts in Figure 5I but marked with arrows) are born isolated and migrate towards each other to assemble a mirror image of the right side group (Figure 5I frames b and c). In the meantime, the pharyngeal precursors move from the ventral surface to the inside of the embryo. This process starts with the MS cells at around 160 minutes pfc (Figure 5I frame a). The AB cells first move on the ventral surface towards the midline (see above) to cover the MS cells (Figure 5I frame b) before following them inside (Figure 5I frame c). This stage of morphogenesis, which we term the assembly stage, ends at ~250 minutes pfc. The end result is a contiguous primordium consisting of two flat sheets of cells and an overall left-right symmetry. This is highlighted in Figure 5IV frame a, which marks the correspondences between pharyngeal cells from symmetric lineages.

Bottom Line: Because our approach is specialized for the characteristics of optically sectioned nuclear images, it can achieve superior accuracy in significantly less time than other approaches.Our approach is fast, accurate, available as open source software and its learned shape model is easy to retrain.As our pharynx development example shows, these characteristics make single cell analysis relatively easy and will enable novel experimental methods utilizing complex data sets.

View Article: PubMed Central - HTML - PubMed

Affiliation: Developmental Biology, Sloan-Kettering Institute, 1275 York Avenue, New York, New York 10065, USA.

ABSTRACT

Background: To exploit the flood of data from advances in high throughput imaging of optically sectioned nuclei, image analysis methods need to correctly detect thousands of nuclei, ideally in real time. Variability in nuclear appearance and undersampled volumetric data make this a challenge.

Results: We present a novel 3D nuclear identification method, which subdivides the problem, first segmenting nuclear slices within each 2D image plane, then using a shape model to assemble these slices into 3D nuclei. This hybrid 2D/3D approach allows accurate accounting for nuclear shape but exploits the clear 2D nuclear boundaries that are present in sectional slices to avoid the computational burden of fitting a complex shape model to volume data. When tested over C. elegans, Drosophila, zebrafish and mouse data, our method yielded 0 to 3.7% error, up to six times more accurate as well as being 30 times faster than published performances. We demonstrate our method's potential by reconstructing the morphogenesis of the C. elegans pharynx. This is an important and much studied developmental process that could not previously be followed at this single cell level of detail.

Conclusions: Because our approach is specialized for the characteristics of optically sectioned nuclear images, it can achieve superior accuracy in significantly less time than other approaches. Both of these characteristics are necessary for practical analysis of overwhelmingly large data sets where processing must be scalable to hundreds of thousands of cells and where the time cost of manual error correction makes it impossible to use data with high error rates. Our approach is fast, accurate, available as open source software and its learned shape model is easy to retrain. As our pharynx development example shows, these characteristics make single cell analysis relatively easy and will enable novel experimental methods utilizing complex data sets.

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