Figure 4

Image analysis of phagocytic cups. (A) Typical fluorescence image of COS-7 cell with FcγR-GFP shown in green and IgG antibodies on 1.5 μ m-radius particles shown in blue. (B, C) Illustrative schematics of 3-dimensional confocal fluorescence microscopy images. (B) Schematic (side view) of a regular cup. (C) Schematic (side view) of a variable cup. From the intersections of the confocal imaging planes with the phagocytic cup, the maximal height of the cell membrane is determined for each angular segment around the particle (red dots). (D) Membrane height as a function of angular segment (see inset in panel F), corresponding to the cup drawn in panel B (dashed line) and C (solid line). Only the angular segments from index 1 to 12 out of 24 are shown. The variability of the cup is defined as the standard deviation divided by the average of this function. The higher this measure, the more variable the cup. (E, F) Side and top views of half-engulfed particles, with cups reconstructed from confocal microscopy data. (E) Top view, taken in the equator plane of the particle, shows a regular distribution of cell membrane (receptors) around the particle (regular cup). (F) Top view shows an irregular distribution of cell membrane (variable cup). Inset Schematic defines the angular segments (in top view).