Counting and measuring IMPs and pits: why accurate counts are exceedingly rare.

Particle counting and measuring techniques are now widely used to characterize normal membranes and to identify molecular changes occurring during development, maturation, and aging during progression of disease and following pharmacological manipulation. However, the use of particle counting and measuring for the identification of molecular changes in membranes has been premature. We show that current procedures rarely yield replicas that are free of cryogenic or mechanical prefractures, and as a result, the "complementarity" of membrane faces is severely compromised. However, with simple alterations of procedure, combined with the resolve to recognize and discard images of pre-fractured membrane faces, a high degree of "complementarity" may be obtained. Criteria for recognizing the occurrence and relative frequency of noncomplementarity are presented and a cleaving method for avoiding a primary source of water vapor contamination is described. In such replicas, membrane pits are found in equivalent numbers and near-identical diameters as the intramembrane particles (IMPs) in the complementary-type membrane faces. When conditions of "cold fracture" and immediate replication are demonstrated, fracture faces are minimally contaminated by frozen water vapor, yielding images where 1) diameters of IMPs vs. pits are very nearly identical, 2) large diameter IMPs are very rare, and 3) the numbers of IMPs and pits are increased substantially over the numbers currently reported. Thus, we reiterate previous proposals that complementarity of membrane faces is the single most important criterion that must be met before accepting the validity of IMP counts or for attributing perceived changes in IMP density or size to conditions of experimental manipulation, to normal developmental processes, or to disease etiology.