Digital photogrammetric quantification of surface area and volume on scanning electron micrographs of frozen hydrated lung tissue

A digital video plotter (DVP, Leica), the personal computer equivalent of an analytical plotter, was used to measure the coordinates of points chosen from stereo pair images of the surface of frozen hydrated lung imaged at magnifications of 2000 and 5000 X with a low-temperature scanning electron microscope (SEM). Rat lung tissue was frozen in vivo with a liquid nitrogen cryoprobe under carefully controlled physiologic conditions. At slow freezing rates, water in the aqueous layer at the surface of the lung segregates into ice crystals (dendrites) which branch in the plane of the surface. Coordinates of points on dendrite surfaces were measured by the DVP and passed to TERRAMODEL (Plus III Software, a land modeling program) where they were used to generate a three-dimensional model, from which surface area and planimetric area of the lung surface were calculated. Additional measurements were made at the top and bottom of the ice structures and a Basic language program was written to calculate the volume of ice on the lung surface. Digital photogrammetry coupled with low-temperature SEM of frozen samples allows measurement of water and water-containing microstructures ubiquitous in biology.