Image processing of immunogold-labeled titin filaments in skeletal muscle

Abstract

In our electron microscopic studies of the elastic behavior of titin filaments in skeletal muscle sarcomeres, immunogold techniques have been used to detect the elastic stretch-response of titin epitopes in the I-band region. The use of gold conjugates was essential, since in highly stretched muscles, distortion of the sarcomere led to the loss of lateral registration of epitopes. In such cases, antibody densities are smeared and difficult to detect. We observed, however, that gold beads sufficiently small ( < 5 nm ) to penetrate the I-band lattice were often obscured by electron dense stains. To overcome this technical problem, we developed a procedure using image processing techniques to differentiate and identify gold beads.In the following example, titin filaments were rendered visible by stretching rabbit semitendinosus muscle using a split-fiber technique to a length where myosin and actin filaments no longer overlap. The titin filaments in the gap region were labeled by monoclonal anti-titin (RT13) followed by protein A-gold conjugates.