(A) When a block of frozen tissue is struck by a knife blade, a fracture plane runs through the tissue, often following a path that leads it through the middle of the lipid bilayer. The fracture plane goes around the proteins rather than cracking them in half, and they segregate with one of the two halves of the bilayer. The exposed faces within the center of the bilayer can then be covered with a metal deposit to form a metallic replica. These exposed faces are referred to as the E, or ectoplasmic face, and the P, or protoplasmic face. (B) Replica of a freeze-fractured human erythrocyte. The P fracture face is seen to be covered with particles approximately 8 nm in diameter. A small ridge (arrow) marks the junction of the particulate face with the surrounding ice. (C) This micrograph shows the surface of an erythrocyte that was frozen and then fractured, but rather than preparing a replica, the cell was thawed, fixed, and labeled with a marker for the carbohydrate groups that project from the external surface of the integral protein glycophorin. Thin sections of the labeled, fractured cell reveal that glycophorin molecules (black particles) have preferentially segregated with the outer half of the membrane. The red line shows the path of the fracture plane.