OOPS: Object-Oriented Polarization Software for analysis of fluorescence polarization microscopy images
Fig 2
Object-based analysis reveals broad biological diversity in desmosomal cadherin architecture.
Desmosomal cadherin order probes expressed in A-431 cells, imaged with FPM, and analyzed with OOPS to demonstrate object-oriented FPM image analysis. (A) Representative intensity (top left) and order (p, top right) images of the Desmocollin 2b extracellular order probe (ECTOb). A single cell-cell border containing several desmosomes is indicated with a rectangular ROI and magnified below. Objects detected after segmentation are enclosed by white boundaries. Two objects that differ in order and signal-to-background ratio (S/B) are indicated by arrows. (B) Closer inspection of the low S/B object in (A). (Top left) Object intensity images at each excitation polarization, normalized to the maximum intensity in the stack. Arrows indicate the direction of the excitation field. (Top right) Average intensity image, normalized to the maximum intensity. Labels indicate regions used to determine local S/B (S: signal; B: background). (Bottom left) Pixel intensity stacks normalized to the total intensity and fit to a generic sinusoid (gray: individual pixel fits; blue: individual pixel azimuths; black: average of all fits). (Bottom right) Object order image. Pixels used to calculate mean order are enclosed in a white boundary. (C) Same as (B), but for the high S/B object indicated in (A). (D) Swarm plots showing mean order () for each object in the desmosome dataset, grouped by construct: ECTOb (red), CYTO (blue), LINK (green), ECTOa (good) (purple), ECTOa (poor) (yellow). (E) Scatter plot of
versus local S/B for each object, grouped as in (D). (F) All objects across all groups were sorted into low and high S/B clusters using k-means clustering. Objects are represented by their average intensity images, which are tiled and stitched together within each cluster: Cluster 1 (light blue; low S/B) and Cluster 2 (dark blue; high S/B). (G) Same as in (D) but grouped by both construct and cluster. (H) Same as in (E) but grouped by cluster.