Correlated Confocal and Intermediate Voltage Electron Microscopy of Myofibrillogenesis in Muscle Cells: Methods and Preliminary Results

Immunofluorescence studies have helped to elucidate the process of molecular assembly into cross-striated myofibrils in cultured cardiac and skeletal muscle cells by localizing accurately identified proteins. However, our understanding of how such immunofluorescence images are related to fine structural organization is limited. High and intermediate voltage electron microscopes can image relatively thick specimens, including whole cells, and confocal scanning laser fluorescence microscopy provides thin optical section images of similarly thick specimens. It seemed natural for us to combine these two approaches in a correlated electron and light microscopic analysis of myofibrillogenesis in cultured cardiac muscle cells.Cardiac myocytes from 7-9 day chick embryos, cultured for 3-5 days on glass coverslips carrying gold EM grids covered with formvar/carbon films, were fixed with 2% formaldehyde for 3 min., permeabilized with 5% Triton for 30 min., and stained with two antibodies conjugated with FITC and rhodamine. These specimens were first observed by epi-fluorescence and then by confocal laser scanning microscopy (Sarastro, Stockholm, SWEDEN).