A simple method for freeze-drying of macromolecules and macromolecular complexes

We present a simple approach for effective freeze-drying and rotary shadowing of large molecules, molecular assemblies, and cell organelles. Simply, a suspension of specimen is adsorped to a glass coverslip, stabilized, and rinsed with 30% methanol. A second coverslip is "sandwiched" on top, and excess methanol is withdrawn from the edges then frozen by plunging into liquid nitrogen and split. Following either rotary or unidirectional shadowing and replication, the coverslip is dissolved in hydrofluoric acid. In addition to avoiding the problems encountered with air-drying specimens for rotary shadowing, the technique also reproducibly provides the thin layer of solution necessary for proper freeze-drying, regardless of how hydrophobic the sample is. The "glass sandwich" technique allows modification of the glass substrate (making it hydrophobic with carbon or hydrophilic by soaking it in alcian blue) which clearly alters the shape of macromolecular assemblies such as myosin filaments and decorated thin filaments.