Membrane allocation profiling: A method to characterize three-dimensional cell shape and attachment based on surface reconstruction

Three-dimensional surface reconstructions from high resolution image stacks of biological specimens, observed by confocal microscopy, have changed the perspective of morphological understanding. In the field of cell–cell or cell–substrate interfaces, combining these two techniques leads to new insights yet also creates a tremendous amount of data. In this article, we present a technique to reduce large, multidimensional data sets from confocal microscopy into one single curve: a membrane allocation profile. Reconstructed cells are represented in a three-dimensional surface from image sections of individual cells. We virtually cut segments of the reconstructed cell membrane parallel to the substrate and calculate the surface areas of each segment. The obtained membrane allocation profiles lead to morphological insights and yield an in vivo ratio of attached and free membrane areas without cell fixation. As an example, glass substrates were modified with different proteins (fibronectin, laminin, concavalin A, extracellular matrix gel, and both isomers of poly-lysine) and presented to HEK293 cells to examine differences in cell morphology and adhesion. We proved that proteins on a substrate could increase the attached portion of a cell membrane, facing the modified substrate, from an average of 32% (glass) to 45% (poly-lysine) of the total membrane surface area.