SAMA: A Method for 3D Morphological Analysis

Abstract

Three-dimensional (3D) culture models are critical tools for understanding tissue morphogenesis. A key requirement for their analysis is the ability to reconstruct the tissue into computational models that allow quantitative evaluation of the formed structures. Here, we present Software for Automated Morphological Analysis (SAMA), a method by which epithelial structures grown in 3D cultures can be imaged, reconstructed and analyzed with minimum human intervention. SAMA allows quantitative analysis of key features of epithelial morphogenesis such as ductal elongation, branching and lumen formation that distinguish different hormonal treatments. SAMA is a user-friendly set of customized macros operated via FIJI (http://fiji.sc/Fiji), an open-source image analysis platform in combination with a set of functions in R (http://www.r-project.org/), an open-source program for statistical analysis. SAMA enables a rapid, exhaustive and quantitative 3D analysis of the shape of a population of structures in a 3D image. SAMA is cross-platform, licensed under the GPLv3 and available at http://montevil.theobio.org/content/sama.

Fig 1. Flow-chart and user-interfaces of SAMA.

General algorithm of functions in SAMA (top panel). The two main interfaces of SAMA—SAMA-Images (bottom left) and SAMA-Analyze (bottom right).

Fig 2. Basic morphometrics, branching and lumen analysis in the 3D hormone-sensitive breast model.

(a) An overview of all 3D structures (top) and individual 3D structure showing budding from E2+Prl (bottom left) and branching from E2+P (bottom right) treatment groups. (b) Branching of structures in E2 alone (top) and E2+Prl (bottom). (c) Raw image of structures with lumena (top) and the processed image of structures with segmented lumena (bottom).

Fig 3. Morphometric analysis of structures in E2+Prl treatment group.

(a) Graphs represent 3D parameters that were calculated using the formula shown in Table 1; elongation (top left), presence of lumen, (top right), sphericity (bottom left) and branching (bottom right). X-axis represents the mean of the parameters and Y-axis represents the distribution of the structures measured in three experimental replicates. Blue (solid) line represents E2 alone treatment and pink (dashed) line represents E2+Prl. (b) Principal component analysis (PCA) shows variance in the data by analyzing correlated variables between E2 and E2+Prl. Factor map with vectors represent highly correlated variables as Dimension 1 and 2 on the x and y axes respectively. Correlated variables sphericity (S) and ratio volume ellipsoid (RVE) in E2 treatment shown with blue arrows while correlated variables elongation (E), number of lumen (L) and complexity (C) in E2+Prl shown with pink arrows (left). Graph shows distribution of the structures in each treatment group, E2 (blue) and E2+Prl (pink) with correlated variables (right).

Fig 4. Morphometric analysis of structures in E2+P treatment group.

(a) Graphs represent 3D parameters that were calculated using the formula shown in Table 1; elongation (top left), elongation in structures with lumen (top right), sphericity (bottom left) and branching in terms of ratio volume ellipsoid (bottom right). X-axis represents the mean of the parameters and Y-axis represents the distribution of the structures measured in three experimental replicates. Blue (solid) line represents E2 alone treatment and pink (dashed) line represents E2+P. (b) Principal component analysis (PCA) shows variance in the data by analyzing correlated variables, sphericity (S), ratio volume ellipsoid (RVE), elongation (E), complexity (C) and number of lumen (L) between E2 and E2+P. Factor map with vectors represent highly correlated variables as Dimension 1 and 2 on the x and y axes respectively. Vector colors, blue, pink and black correspond to the trend in the distribution of structures in E2, E2+P and both respectively (left). Graph shows distribution of the structures in each treatment group, E2 (blue) and E2+P (pink) with correlated variables (right). (c) Graphs represent reproducibility in the measurement of elongation in structures of E2 (left) and E2+P (right). X-axis represents the mean of the parameters and y-axis represents the distribution of the structures measured in five experimental replicates shown as a number code.