FIG. 5.

Object identification and multiparameter analysis in the PLN. (a) Sample of a data analysis pipeline identifying DCs in contact with collagen within the PLN. (i) Raw image is loaded into the CellProfiler pipeline; (ii) Image is separated into its component channels, and DCs are identified using pre-defined algorithms; (iii) DCs are analyzed for multiple parameters, and are gated on co-localization of the DC perimeter with collagen signal; (iv) DCs are sorted based on the displayed gate, and are visually classified as collagen-associated DCs (green) or as nonassociated DCs (blue) superimposed on the original collagen channel image. (b) Sample of combinatorial analysis from multiple XY planes showing DC form factor (a measure of the spherical nature of each object) as a function of collagen association. (i) Scatter plot of the analysis for a single XY plane, showing DC form factor as a function of collagen association. (ii) Combined data from (3) XY planes, representing 150 μm of contiguous imaging data from (1) PLN gated on collagen associated DCs. (iii) DC form factor analysis from (ii) of collagen associated vs. nonassociated DCs. A color version of this figure is available in the online article at www.liebertpub.com/lrb