National Institute of Standards and Technology

3D Image Segmentation

Summary

Three-dimensional (3D) image segmentation is encountered in scientific studies of z-stacks acquired by confocal laser scanning microscopes. Our objectives are (a) to automate segmentation of a large number of 3D z-stacks and (b) to estimate the segmentation accuracy from projected ground truth, statistical samples and visual inspection inputs.

3D image segmentation of human bone marrow stromal cell cultured in Collagen Gel scaffold. (1) XY projection of the 3D image acquired  using confocal laser scanning microscopy. (2) XY projection of the 3D image obtained after 3D segmentation. (3) Segmented 3D image volume observed in the 3D interactive viewer.
3D image segmentation of human bone marrow stromal cell cultured in Collagen Gel scaffold. Top left (1): XY projection of the 3D image acquired using confocal laser scanning microscopy. Top right (2): XY projection of the 3D image obtained after 3D segmentation. Bottom (3): Segmented 3D image volume observed in the 3D interactive viewer.

Description

Human bone marrow stromal cells were cultured on 10 different scaffolds and imaged in 3D using confocal laser scanning microscopy. Approximately 110 cells were imaged per scaffold yielding 1100 Z-stacks, 122,949 images and 122 GB of data. Six segmentation processes were designed and the most accurate method was selected upon imaging and geometrical assumptions. Selection was driven by minimization of human labor needed to provide ground truth while maximizing statistical confidence in the segmentation accuracy.

Segmentation accuracy was determined against manually segmented z-stacks. The two most accurate segmentation methods were applied to all z-stacks and visually inspected in a 2D tiled view and interactive 3D view in a web browser. The results of visual inspections are incorporated into the analyses to increase the confidence in segmentation accuracy estimates.

Online 3D visualization tool

We built a 3D web-based visualization tool allowing the visualization of 3D volumes representing the segmented z-stacks.

A 3D web-based visualization of 100+ z-stacks from the same 
                             collagen scaffold type.
A 3D web-based visualization of 100+ z-stacks from the Collagen Gel scaffold type. The insets illustrate the interactivity during visual inspection. The blue ball is used as a spatial scale.

This tool is accessible online by clicking on the following link:

Online 3D visualization tool

The 3D shapes obtained from actin or nucleus segmented images are displayed by selecting scaffolds of interest or individual cells on the top-left menu and clicking on the "Load Meshes" button. Different color-coding and sorting options are also available on the bottom-left menu.

A rapid cell shape comparison between several scaffolds can be easily performed since the tool is able to load all the meshes at the same time at a lower-resolution. More detailed analysis of a particular mesh is also possible by right clicking on a cell of interest which will load the 3D volume at a full resolution. Zoom-in and out are enabled through the mouse-wheel and the "+" and "-" keys. Further help is available by clicking on the "?" button next to the "Load Meshes" button.

Explanation of Color/Order Metrics:

- File Name: file name
- Volume: 3D cell volume
- Surface area: 3D cell surface area
- Z-Depth: 3D cell volumes were fit with a gyration tensor and the "square roots of the principal moments of the gyration tensor" denoted the semi- axis lengths [sqrt(L1), sqrt(L2), sqrt(L3)] of a characteristic ellipsoid that was representative of each cell. Z-Depth is the caliper- diameter parallel to the smallest axis [sqrt(L1)]. The caliper diameter, also known as the Feret diameter, is the size of an object along a specific direction.
- Sqrt(L1): 3D cell volumes were fit with a gyration tensor and the “square roots of the principal moments of the gyration tensor” denoted the semi-axis lengths of a characteristic ellipsoid that was representative of each cell. Sqrt(L1) is the length of the shortest semi-axis of the characteristic ellipsoid.
- Sqrt(L2): 3D cell volumes were fit with a gyration tensor and the “square roots of the principal moments of the gyration tensor” denoted the semi-axis lengths of a characteristic ellipsoid that was representative of each cell. Sqrt(L2) is the length of the median- length semi-axis of the characteristic ellipsoid.
- Sqrt(L3): 3D cell volumes were fit with a gyration tensor and the “square roots of the principal moments of the gyration tensor” denoted the semi-axis lengths of a characteristic ellipsoid that was representative of each cell. Sqrt(L3) is the length of the longest semi-axis of the characteristic ellipsoid.

The raw z-stacks, volumetric and mesh representations of segmentation results, and 3D shape features are accessible from the following link:

Download web page for raw and processed z-stacks

Date created: April 10, 2014 | Last updated: