Elastic Volume Reconstruction from Series of Ultra-thin Microscopy Sections

Stephan Saalfeld, Richard Fetter, Albert Cardona & Pavel Tomancak

Supplementary Videos

Supplementary Video 1

Visualization of the behavior of spring meshes during elastic alignment. The movie shows the iterative relaxation of the spring-particle system for five examplary serial sections from the C. elegans series. Sections were pre-aligned rigidly such that all section spring-meshes start from the same initial position. Zero-length cross-section springs connect all vertices to their corresponding location in other sections (except for those where no match could be found by block matching). Springs are displayed as lines with their color reflecting the relative stress applied. The color ranges from green (fully relaxed) to red (maximally stressed) and is scaled relative to the maximum stress present in the system. Initially, all intra-section-mesh springs are green and all cross-section springs are maximally stressed. After relaxation, stresses are distributed throughout the system. Cross-section springs have contracted and the series is warped into alignment.

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Supplementary Video 2

Evaluation Series A. The movie shows Evaluation Series A. The series consists of 400 sections of 2,000×2,000px each, all sections showing the same artificially generated image. The original series has been deformed non-linearly and then aligned using a rigid or affine model per section (rigid, affine) and using our elastic method (elastic).

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Supplementary Video 3

Evaluation Series B. The movie shows Evaluation Series B. The series consists of 400 sections from an artificially generated volume of 2,000×2,000×800px. The section thickness is 2px. The original series has been deformed non-linearly and then aligned using a rigid or affine model per section (rigid, affine) and using our elastic method (elastic).

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Supplementary Video 4

Walk through the rigidly aligned C. elegans series. The movie shows in each frame a single section from the rigidly aligned TEM section series of a C. elegans three-fold stage embryo at four different zoom levels (A-D where B shows the close up of the blue rectangled area in A etc.). The scale bars in each panel are 4 µm (A), 2 µm (B), 1 µm (C) and 0.5 µm (D). The lateral resolution of the raw data is 4 nm/pixel. The number in the upper right corner indicates the section index. Note the significant non-linear distortion remaining in the rigidly aligned series.

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Supplementary Video 5

Walk through the rigidly aligned C. elegans series orthogonally re-sliced. The movie shows in each frame a single, computationally generated section orthogonal to the physical sectioning plane, through the rigidly aligned TEM section series of a C. elegans three-fold stage embryo. Sections were down-sampled such that their lateral resolution matches the section thickness of 50 nm/pixel. Accordingly, orthogonal sections are generated at a step-size of 50 nm. The number in the upper right corner indicates the virtual section index. Two different zoom levels are shown (A,B) where B corresponds to the blue rectangle in A. The scale bars in the panels are 5 µm (A) and 2 µm (B). Note the strong non-linear distortion remaining in the rigidly aligned series that effectively prevents identification of biologically relevant features in the axial direction.

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Supplementary Video 6

Walk through the elastically aligned C. elegans series. The movie shows in each frame a single section from the elastically aligned TEM section series of a C. elegans three-fold stage embryo at four different zoom levels (A-D where B shows the close up of the blue rectangled area in A etc.). The scale bars in each panel are 4 µm (A), 2 µm (B), 1 µm (C) and 0.5 µm (D). The lateral resolution of the raw data is 4 nm/pixel. The number in the upper right corner indicates the section index. Note the noticeable improvement in amount of non-linear distortion remaining in the elastically aligned series compared to the rigidly aligned series (Supplementary Video 4). The data can be interactively browsed in CATMAID.

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Supplementary Video 7

Walk through the elastically aligned C. elegans series orthogonally re-sliced. The movie shows in each frame a single, computationally generated section, orthogonal to the physical sectioning plane, through the elastically aligned TEM section series of a C. elegans three-fold stage embryo. Sections were down-sampled such that their lateral resolution matches the section thickness of 50 nm/pixel. Accordingly, orthogonal sections are generated at a step-size of 50 nm. The number in the upper right corner indicates the virtual section index. Two different zoom levels are shown (A,B) where B corresponds to the blue rectangle in A. The scale bars in the panels are 5 µm (A) and 2 µm (B). Note that the data have a familiar TEM appearance in the axial direction owing to the near complete removal of non-linear distortion by the elastic alignment method.

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Supplementary Video 8

Walk through the rigidly aligned Drosophila series. The movie shows in each frame a single section from the rigidly aligned TEM section series, imaged as mosaic of overlapping tiles, of the D. melanogaster first instar larval ventral nerve cord at four different zoom levels (A-D where B shows the close up of the blue rectangled area in A etc.). The scale bars in each panel are 10 µm (A), 3 µm (B), 1 µm (C) and 0.5 µm (D). The lateral resolution of the raw data is 4 nm/pixel. The number in the upper right corner indicates the section index. Note the significant non-linear distortion remaining in the rigidly aligned data.

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Supplementary Video 9

Walk through the rigidly aligned Drosophila series orthogonally resliced. The movie shows in each frame a single, computationally generated section, orthogonal to the physical sectioning plane, through the rigidly aligned TEM section series of the D. melanogaster first instar larval ventral nerve cord imaged as mosaic of overlapping tiles. Sections were down-sampled such that their lateral resolution matches the section thickness of 45 nm/pixel. Accordingly, orthogonal sections are generated at a step-size of 45 nm. The number in the upper right corner indicates the virtual section index. Two different zoom levels are shown (A,B) where B corresponds to the blue rectangle in A. The scale bars in the panels are 5 µm (A) and 3 µm (B). The lateral resolution of the raw data is equal to the section thickness that is 45 nm/pixel. Note the strong non-linear distortion remaining in the rigidly aligned data that effectively prevents identification of biologically relevant features in the axial direction.

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Supplementary Video 10

Walk through the elastically aligned Drosophila series. The movie shows in each frame a single section from the elastically aligned TEM section series, imaged as mosaic of overlapping tiles, of the D. melanogaster first instar larval ventral nerve cord at four different zoom levels (A-D where B shows the close up of the blue rectangled area in A etc.). The scale bars in each panel are 10 µm (A), 3 µm (B), 1 µm (C) and 0.5 µm (D). The lateral resolution of the raw data is 4 nm/pixel. The number in the upper right corner indicates the section index. Note the noticeable improvement in amount of non-linear distortion remaining in the elastically aligned series compared to the rigidly aligned series (Supplementary Video 8).

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Supplementary Video 11

Walk through the elastically aligned Drosophila series orthogonally resliced. The movie shows in each frame a single, computationally generated section, orthogonal to the physical sectioning plane, through the elastically aligned TEM section series of the D. melanogaster first instar larval ventral nerve cord imaged as mosaic of overlapping tiles. Sections were down-sampled such that their lateral resolution matches the section thickness of 45 nm/pixel. Accordingly, orthogonal sections are generated at a step-size of 45 nm. The number in the upper right corner indicates the virtual section index. Two different zoom levels are shown (A,B) where B corresponds to blue rectangle in A. The scale bars in the panels are 5 µm (A) and 3 µm (B). Note that the data have a familiar TEM appearance in the axial direction owing to the near complete removal of non-linear distortion by the elastic alignment method.

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Supplementary Video 12

Rigid Alignment of an Array Tomography Series. The movie shows a sample series of 43 serial sections of 70 nm thickness each from barrel cortex of an adult Line H YFP mouse18 expressing YFP in a subset of layer 5b pyramidal cells, pial surface at the top. Each section shows three fluorescent channels imaged by light microscopy as an aribitrary RGB overlay, DAPI (blue), YFP (green) and Synapsin (red). As with ssTEM data there is significant remaining non-linear distortion present in the rigidly aligned dataset. Data courtesy of Forrest Collman, Nick Weiler, Kristina Micheva, and Stephen Smith.

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Supplementary Video 13

Elastic Alignment of an Array Tomography Series. The movie shows a sample series of 43 serial sections of 70 nm thickness each from barrel cortex of an adult Line H YFP mouse18 expressing YFP in a subset of layer 5b pyramidal cells, pial surface at the top. Each section shows three fluorescent channels imaged by light microscopy as an aribitrary RGB overlay, DAPI (blue), YFP (green) and Synapsin (red). Analogously to ssTEM data, the elastic alignment removes most of the non-linear distortion from the light microscopy section series. Data courtesy of Forrest Collman, Nick Weiler, Kristina Micheva, and Stephen Smith.

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Supplementary Video 14

3d rendering of elastically aligned Array Tomography Series. Part of the elastically aligned dataset shown in Supplementary Video 13 was used to generate a 3d volume rendering that demonstrates how well neuronal morphology can be reconstructed from the elastically aligned series. Data courtesy of Forrest Collman, Nick Weiler, Kristina Micheva, and Stephen Smith.

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