FIMD analysis compares the fluorescence intensity at the entrance of a micropipette with the intensity at the cap. (A) Simulation of an aspirated RBC reference clearly shows the spectrin-actin network to be compressed at the entrance, e, and dilated at the cap, c. Membrane proteins associated with the spectrin-actin cytoskeleton such as glycophorin C show fluorescence profiles reflecting these network gradients. (B) Glycophorin C shows only a slight change in projection gradient or fitted slope for normal versus 4.2-Nippon (4.2-N) cells; asterisks indicate statistically different slopes. In contrast, CD47 on 4.2-deficient cells shows a significant and clear reduction in connectivity from the normal.