Analysis of spatial distribution of marker expression in cells using boundary distance plots

Boundary distance (BD) plotting is a technique for making orientation invariant comparisons of the spatial distribution of biochemical markers within and across cells/nuclei. Marker expression is aggregated over points with the same distance from the boundary. We present a suite of tools for improved data analysis and statistical inference using BD plotting. BD is computed using the Euclidean distance transform after presmoothing and oversampling of nuclear boundaries. Marker distribution profiles are averaged using smoothing with linearly decreasing bandwidth. Average expression curves are scaled and registered by x-axis dilation to compensate for uneven lighting and errors in nuclear boundary marking. Penalized discriminant analysis is used to characterize the quality of separation between average marker distributions. An adaptive piecewise linear model is used to compare expression gradients in intra, peri and extra nuclear zones. The techniques are illustrated by the following: (a) a two sample problem involving a pair of voltage gated calcium channels (Cav1.2 and AB70) marked in different cells; (b) a paired sample problem of calcium channels (Y1F4 and RyR1) marked in the same cell.