Stereometric estimation of the area of the freeze‐fractured membrane

Stereo‐pair micrographs of freeze‐fracture replicas taken at +6 and −6 degrees and enlarged 55,000 diameters were precisely aligned on a digitizing pad linked to a plotter and programmable calculator. A point grid spaced at 0.23 μm was placed over the left photo‐pair. Under stereoscopic observation, matching points were placed by hand on the right photo‐pair for every point in the applied grid that fell over the membrane surface of interest in the left photo‐pair. The x, y coordinates of every point in the two photos were read with the digitizer. These data allowed the calculation of the X, Y, Z coordinates of all points in the untilted object space, 3‐D reconstruction of the analyzed surface, and estimation of the membrane surface area.