Bidirectional shadowing in freeze-etching.

Bidirectional shadowing in freeze-etching may be achieved by firing an electron-beam shadowing source, rotating the specimen stage through a desired angle, and re-firing the shadowing source. It is demonstrated that portrait shadow-casting, which permits information to be drawn from much of the specimen region lying within primary shadows, can be readily achieved using a 90 degree specimen rotation. With 180 degree specimen rotation, particle-size analysis is feasible. Particle-height analysis is demonstrated using membrane-associated particles as an example. Data from suitable sets of micrographs can also be used for the estimation of particle-width exaggeration due to the accumulation of the shadowing-metal cap. Fibre-width analysis, using the linear regression method, is demonstrated by a study of native cellulosic microfibrils. Mean microfibril widths were found to be 5.5-7.0 nm.